Evidence of novel type of ribosome in eukaryotic intermediate flatworm

In all organisms, messenger-directed protein synthesis is catalyzed by ribonucleoprotein particles called ribosomes. A ribosome is typically composed of one small and one large subunit which contain one short (18S) and one long (28S) rRNAs, respectively. Surprisingly, in this study, three similar size rRNAs (18-21S) were revealed in the electrophoresis profile of the total RNAs of tapeworm _Spirometra erinaceiuropaei_. Northern blot analysis shows that one of the three bands belongs to 18S rRNA, and the other two bands are of 28S rRNAs, implying structurally distinct ribosomes in this intermediate animal. Furthermore, similar, but not identical profiles were observed in two other tapeworms _Diphyllobothrium hottai_ and _Diphyllobothrium Nipponkaiizeme_. Relevant to this finding, in flatworm _Paragonimus westermani_, 18S rRNAs were found much more numerous than 28S rRNAs. Moreover, consistent with this biochemical finding, transmission electron microscopy examinations show that the ribosomes isolated from _Spirometra erinaceiuropaei_ are composed of either one ball or two similar size subunits (balls), while the structure of ribosomes isolated from control liver tissue exactly match the conventional large and small subunit ribosome model. Our study provides direct biochemical and biophysical evidence of structurally distinct novel type of ribosomes in intermediate eukaryotic flatworms. These finding may be important for re-recognition of biological protein synthesis and evolutionary process of living things

Broad application of a simple and affordable protocol for isolating plant RNA

BACKGROUND: Standard molecular biological methods involve the analysis of gene expression in living organisms under diverse environmental and developmental conditions. One of the most direct approaches to quantify gene expression is the isolation of RNA. Most techniques used to quantify gene expression require the isolation of RNA, usually from a large number of samples. While most published protocols, including those for commercial reagents, are either labour intensive, use hazardous chemicals and/or are costly, a previously published protocol for RNA isolation in Arabidopsis thaliana yields high amounts of good quality RNA in a simple, safe and inexpensive manner. FINDINGS: We have tested this protocol in tomato and wheat leaves, as well as in Arabidopsis leaves, and compared the resulting RNA to that obtained using a commercial phenol-based reagent. Our results demonstrate that this protocol is applicable to other plant species, including monocots, and offers yield and purity at least comparable to those provided by commercial phenol-based reagents. CONCLUSIONS: Here, we show that this previously published RNA isolation protocol can be easily extended to other plant species without further modification. Due to its simplicity and the use of inexpensive reagents, this protocol is accessible and affordable and can be easily implemented to work on different plant species in laboratories worldwide

A Polymerase-chain-reaction Assay for the Specific Identification of Transcripts Encoded by Individual Carcinoembryonic Antigen (CEA)-gene-family Members

Carcinoembryonic antigen (CEA) is a tumor marker that belongs to a family of closely related molecules with variable expression patterns. We have developed sets of oligonucleotide primers for the specific amplification of transcripts from individual CEA-family members using the reverse transcriptase/ polymerase chain reaction (RT/PCR). Specific primer sets were designed for CEA, non-specific cross-reacting antigen (NCA), biliary glycoprotein (BGP), carcinoembryonic antigen gene-family members 1, 6 and 7 (CGMI, CGM6 and CGM7), and one set for all pregnancy-specific glycoprotein (PSG) transcripts. Primers were first tested for their specificity against individual cDNA clones and product-hybridization with internal, transcript-specific oligonucleotides. Total RNA from 12 brain and 63 gynecological tumors were then tested for expression of CEA-related transcripts. None were found in tumors located in the brain, including various mesenchymal and neuro-epithelial tumors. CEA and NCA transcripts were, however, present in an adenocarcinoma located in the nasal sinuses. In ovarian mucinous adenocarcinomas, we always found co-expression of CEA and NCA transcripts, and occasionally BGP mRNA. CEA-related transcripts were also found in some serous, endometrioid and clear-cell ovarian carcinomas. CEA, NCA and BGP transcripts were present in endometrial carcinomas of the uterus and cervical carcinomas, whereas uterine leiomyomas were completely negative. No transcripts were found from CGM 1, CGM6, CGM7 or from PSG genes in any of the tumors tested. The PCR data were compared with immunohistochemical investigations of ovarian tumors at the protein level using CEA (26/3/13)-, NCA-50/90 (9A6FR) and NCA-95 (80H3)-specific monoclonal antibodies

A new robust diagnostic polymerase chain reaction for determining the mating status of female Anopheles gambiae mosquitoes.

The principal malaria vector in Africa, Anopheles gambiae, contains two pairs of autosomes and one pair of sex chromosomes. The Y chromosome is only associated with males and other Y chromosome-specific DNA sequences, which are transferred to women during mating. A reliable tool to determine the mating status of dried wild An. gambiae females is currently lacking. DNA was extracted from dried virgin and mated females and used to test whether Y chromosome-specific polymerase chain reaction (PCR) markers can be successfully amplified and used as a predictor of mating. Here we report a new PCR-based method to determine the mating status among successfully inseminated and virgin wild An. gambiae females, using three male-specific primers. This dissection-free method has the potential to facilitate studies of both population demographics and gene flow from dried mosquito samples routinely collected in epidemiologic monitoring and aid existing and new malaria-vector control approaches

Combining mammaglobin and carcinoembryonic mRNA markers for early detection of micrometastases from breast cancers - a molecular study of 59 patients

Introduction: As many as 30% of node-negative breast cancer patients relapse within five years, suggesting that current histological detection methods are inadequate for identifying metastatic disease. Detecting small number of cancer cells in the breast tissue or lymph node by reverse transcription-polymerase chain reaction (RT-PCR) assays using a combination of tissue and cancer specific markers might be very useful in the early detection or monitoring of the treatment. Mammaglobin is a member of the uteroglobin gene family and appears to be expressed only in breast tissue. Carcinoembryonic antigen has been the preferred molecular marker for detection of micro metastases in lymph nodes in almost all carcinomas. Materials and Methods: Samples were collected from randomly chosen breast cancer patients undergoing modified mastectomy or breast conserving surgery between September 2003 and July 2004. RT-PCR was applied to study the expression of MMG and CEA markers. Breast cancer micrometastases in axillary lymph nodes were also assessed. Results: The MMG marker was positive in 9/10 normal breast tissues, 3/ 3 breast fibroadenomas and 37/39 of breast carcinoma tissues, giving an overall sensitivity of 94%. The sensitivity was 80% for metastatic lymph node samples. On the other hand CEA showed 95% sensitivity for malignant breast tumors and 100% sensitivity for metastatic lymph nodes. Conclusions: RT-PCR using a combination of MMG and CEA markers is a powerful tool to complement current routine histopathology techniques for detection of breast cancer metastasis in axillary nodes

Isolation and characterization of microsatellites in the lichen Buellia frigida (Physciaceae), an Antarctic endemic

Premise of the study: Microsatellite markers were characterized for an Antarctic endemic, Buellia frigida, to investigate population structure and origin of Antarctic lichens. Methods and Results: Five primer sets were characterized. All loci were polymorphic with eight to 16 alleles per locus in a sample of 59 lichens. Conclusions: The microsatellite markers potentially provide insight into population structure and gene flow of B. frigida

A Rapid and Efficient Method for Purifying High Quality Total RNA from Peaches (Prunus persica) for Functional Genomics Analyses

http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602005000100010&lng=es&nrm=isoPrunus persica has been proposed as a genomic model for deciduous trees and the Rosaceae family. Optimized protocols for RNA isolation are necessary to further advance studies in this model species such that functional genomics analyses may be performed. Here we present an optimized protocol to rapidly and efficiently purify high quality total RNA from peach fruits (Prunus persica). Isolating high-quality RNA from fruit tissue is often difficult due to large quantities of polysaccharides and polyphenolic compounds that accumulate in this tissue and co-purify with the RNA. Here we demonstrate that a modified version of the method used to isolate RNA from pine trees and the woody plant Cinnamomun tenuipilum is ideal for isolating high quality RNA from the fruits of Prunus persica. This RNA may be used for many functional genomic based experiments such as RT-PCR and the construction of large-insert cDNA libraries

The Putative Role of Human Peritoneal Adipocytes in the Fight against Bacteria: Synthesis of the Antimicrobial Active Peptide DEFA1-3

Background: Spontaneous peritonitis is a rather rare event, even in peritoneal dialysis (PD). As defensins are natural antimicrobial peptides, we hypothesized that adipocytes as the major constituents of the omentum could play an important role in protecting against infection by generating defensin (DEFA1-3). Methods: We isolated adipocytes from the omentum majus and conducted qualitative and quantitative RT-PCR and immunohistochemical analyses. Results: For the first time described, we were able to detect DEFA1-3 mRNA in highly purified isolated omental adipocytes. The expression of DEFA1-3 in adipocytes was confirmed on the protein level by immunohistochemistry. Conclusion: Our report of DEFA1-3 expression by human omental adipocytes adds to the role of adipocytes in the primary defense against bacterial infection. This may include PD, where the presence of the catheter as a foreign body and the nonphysiological dialysis solution may require constant defense measures to prevent peritonitis, a hypothesis that will require further testing. Copyright (C) 2010 S. Karger AG, Base

Effect of high glucose concentration on the synthesis of monocyte chemoattractant protein-1 in human peritoneal mesothelial cells: Involvement of protein kinase C

Human peritoneal mesothelial cells (HMC) contribute to the activation and control of inflammatory processes in the peritoneum by their potential to produce various inflammatory mediators. The present study was designed to assess the effect of glucose, the osmotic active compound in most commercially available peritoneal dialysis fluids, on the synthesis of the C-C chemokine monocyte chemoattractant protein-1 (MCP-1) in cultured HMC. The MCP-1 concentration in the cell supernatants was determined by enzyme-linked immunosorbent assay and the MCP-1 mRNA expression was examined using Northern blot analysis. Incubation of HMC with glucose (30-120 mM) resulted in a time- and concentration-dependent increase in MCP-1 protein secretion and mRNA expression. After 24 h the MCP-1 synthesis was increased from 2.8 +/- 0.46 to 4.2 +/- 0.32 ng/10(5) cells (n = 5, p 2001 S. Karger AG. Basel

Transcription of brain natriuretic peptide and atrial natriuretic peptide genes in human tissues

We have compared the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes in various human tissues using a quantitative polymerase chain reaction technique. Tissues of three human subjects, obtained at autopsy, were analyzed. BNP transcripts could be detected in the central nervous system, lung, thyroid, adrenal, kidney, spleen, small intestine, ovary, uterus, and striated muscle. ANP transcripts could also be demonstrated in various human extracardiac tissues including several endocrine organs. In all peripheral tissues, the level of both natriuretic peptide transcripts was approximately 1-2 orders of magnitude lower than in cardiac ventricular tissues. This distribution is in marked contrast to the much lower level of ANP and BNP transcripts present in extracardiac rat tissues (generally less than 1/1000 of ventricles). These data suggest differential expression of the two natriuretic peptide genes in cardiac and extracardiac tissues in man. Furthermore, the presence of local synthesis of ANP and BNP in various peripheral organs suggests paracrine and/or autocrine function of these natriuretic peptides