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At least 1,207 records · Page 67Linked to original sources
PubMed2001

Spatial learning induces neurotrophin receptor and synapsin I in the hippocampus.

We report that rats learning a spatial memory task in the Morris water maze show elevated expression of the signal transduction receptor for BDNF and the synaptic associated protein synapsin I in the hippocampus. Nuclease protection assays showed maximal levels of TrkB and synapsin I mRNAs in the hippocampus by the time that asymptotic learning performance had been reached (Day 6). Increases in synapsin I mRNA were matched by changes in synapsin I protein as revealed by western blot analysis. Synapsin I is a downstream effector for the BDNF tyrosine kinase cascade pathway which has important roles in synaptic remodeling and function. Therefore, parallel changes in TrkB and synapsin I mRNAs suggest a role of the BDNF system in synaptic function or adaptation. Levels of TrkB mRNA in the hippocampus were attenuated after learning acquisition (Day 20), but synapsin I mRNA was still elevated, suggesting that the BDNF system may participate in events secondary to learning, such as strengthening of neural circuits. TrkB and synapsin I mRNAs showed an increasing trend in the cerebellum of learning rats and no changes were observed in the caudal cerebral cortex. The selectivity of the changes in trkB and synapsin I, affecting the hippocampus, is in agreement with the role of this structure in processing of spatial information. Behavioral regulation of neurotrophins may provide a molecular basis for the enhanced cognitive function associated with active lifestyles, and guide development of strategies to promote neural healing after CNS injury or disease.

Animals↗
PubMed2004

Growth patterns reflect response to antiretroviral therapy in HIV-positive infants: potential utility in resource-poor settings.

Laboratory monitoring of HIV-infected children is the current standard of care in the United States to guide the appropriate use of antiretroviral therapy (ART). Although ART is becoming a reality in some developing countries, laboratory monitoring of ART is costly, necessitating creative approaches to monitoring. As an initial step to guide monitoring of HIV progression in low resource settings, we assessed the utility of the physical examination to predict clinical progression of HIV. We conducted a retrospective cohort study of HIV-infected children using data from Pediatric AIDS Clinical Trials Group Protocol 300. We developed a clinical predictive model, and compared the utility of the clinical model to the change in HIV RNA viral load as diagnostic tests of ART failure. The clinical model incorporated treatment regimen, age, and height velocity: a three-level clinical predictive model provided likelihood ratios of 0.3, 3.9, and 14. For decline in RNA the likelihood ratios were 0.2 (> 1 log decline), 1.4, and 3.5 (> log increase). We developed a simple clinical predictive model that was able to predict clinical progression of HIV after initiation of new ART. The clinical model performed similarly to using changes in HIV RNA viral load. These data should be validated internationally and prospectively, because the test subjects were from a resource rich environment and growth patterns in undernourished children may be impacted differently by HIV and its treatment. The model was most pertinent to children 36 months of age or younger, and was conducted in children receiving monotherapy and dual therapy.

Anti-HIV Agents↗
PubMed2026

Target site selection and P1 engineering enable highly efficient circular RNA production via end-to-end self-targeting and splicing.

Circular RNAs (circRNAs) are more stable than linear RNAs, enabling expanding applications in RNA vaccines and therapeutics. We previously developed an in vitro circRNA preparation method based on end-to-end self-targeting and splicing (STS) using the Tetrahymena group I intron, which generates circRNAs without extraneous sequences. However, self-circularization efficiency declines as gene of interest (GOI) length increases, limiting its application to longer GOIs. Here, we systematically optimized key determinants of STS efficiency, including target site selection and P1 construct engineering. Target site screening revealed that selection of optimal target sites within each GOI markedly improved self-circularization efficiency. Moreover, engineering of the P1 construct, including incorporation of a polyA10 sequence upstream of the internal guide sequence of the intron and an antisense sequence complementary to the target site and its upstream region at the 5' side of polyA10, further enhanced efficiency. Notably, the optimized STS strategy achieved up to two-fold higher self-circularization efficiency than the conventional permuted intron-exon (PIE) method for long GOIs (∼8 K-nt). Collectively, these results establish an improved STS workflow for efficient circRNA production without extraneous sequences across a wide range of GOI lengths, outperforming the PIE method for long GOIs, and broadening biomedical applications.

RNA, Circular↗
PubMed2004

Tissue microarrays: a practical guide.

Tissue microarrays are a recent innovation in the field of pathology. They were originally designed as a high-throughput approach for researchers to assess the expression of interesting candidate disease-related genes or gene products simultaneously on hundreds of tissue samples. However, their use is becoming more widespread in routine pathology, for example for quality assurance and for the optimisation of diagnostic reagents such as monoclonal antibodies and gene probes. Several molecular and conventional pathological techniques can be performed on a single tissue array, thereby enabling morphology, DNA, RNA and protein targets to be analysed on sequential sections through multiple tissue samples. Moreover, compared with full-face tissue sections, tissue microarrays are a cost- and time-efficient, effective approach to analysing biomarker expression on a large number of samples. Whilst tissue microarrays are available from commercial sources, many pathology laboratories prefer to make in-house arrays from their often extensive pathology archive to facilitate the correlation of their findings with clinical parameters. The technical skills necessary to produce tissue arrays are well within the capacity of most laboratories. However, several pitfalls to successful array production exist. The present article describes the applications of this technique and details practical points for optimal tissue array production.

Gene Expression Profiling↗
PubMed1997

Viral dynamics of primary viremia and antiretroviral therapy in simian immunodeficiency virus infection.

Mathematical modeling of viral replication dynamics, based on sequential measurements of levels of virion-associated RNA in plasma during antiretroviral treatment, has led to fundamental new insights into human immunodeficiency virus type 1 pathogenesis. We took advantage of the simian immunodeficiency virus (SIV)-infected macaque model to perform detailed measurements and mathematical modeling during primary infection and during treatment of established infection with the antiretroviral drug (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). The calculated clearance half-life for productively infected cells during resolution of the peak viremia of primary infection was on the order of 1 day, with slightly shorter clearance half-lives calculated during PMPA treatment. Viral reproduction rates upon discontinuation of PMPA treatment after 2 weeks were approximately twofold greater than those obtained just prior to initiation of treatment in the same animals, likely reflecting accumulation of susceptible target cells during treatment. The basic reproductive ratio (R0) for the spread of SIV infection in vivo, which represents the number of productively infected cells derived from each productively infected cell at the beginning of infection, was also estimated. This parameter quantifies the extent to which antiviral therapy or vaccination must limit the initial spread of virus to prevent establishment of chronic disseminated infection. The results thus provide an important guide for efforts to develop vaccines against SIV and, by extension, human immunodeficiency virus.

AIDS Vaccines↗
PubMed2004

The design of a potent inhibitor of the hepatitis C virus NS3 protease: BILN 2061--from the NMR tube to the clinic.

The virally encoded serine protease NS3/NS4A is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. Until very recently, the design of inhibitors for the HCV NS3 protease was limited to large peptidomimetic compounds with poor pharmacokinetic properties, making drug discovery an extremely challenging endeavor. In our quest for the discovery of a small-molecule lead that could block replication of the hepatitis C virus by binding to the HCV NS3 protease, the critical protein-polypeptide interactions between the virally encoded NS3 serine protease and its polyprotein substrate were investigated. Lead optimization of a substrate-based hexapeptide, guided by structural data, led to the understanding of the molecular dynamics and electronic effects that modulate the affinity of peptidomimetic ligands for the active site of this enzyme. Macrocyclic beta-strand scaffolds were designed that allowed the discovery of potent, highly selective, and orally bioavailable compounds. These molecules were the first HCV NS3 protease inhibitors reported that inhibit replication of HCV subgenomic RNA in a cell-based replicon assay at low nanomolar concentrations. Optimization of their biopharmaceutical properties led to the discovery of the clinical candidate BILN 2061. Oral administration of BILN 2061 to patients infected with the hepatitis C genotype 1 virus resulted in an impressive reduction of viral RNA levels, establishing proof-of-concept for HCV NS3 protease inhibitors as therapeutic agents in humans.

Carbamates↗
PubMed2004

Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana.

MicroRNAs (miRNAs) in plants and animals function as post-transcriptional regulators of target genes, many of which are involved in multicellular development. miRNAs guide effector complexes to target mRNAs through base-pair complementarity, facilitating site-specific cleavage or translational repression. Biogenesis of miRNAs involves nucleolytic processing of a precursor transcript with extensive foldback structure. Here, we provide evidence that genes encoding miRNAs in plants originated by inverted duplication of target gene sequences. Several recently evolved genes encoding miRNAs in Arabidopsis thaliana and other small RNA-generating loci possess the hallmarks of inverted duplication events that formed the arms on each side of their respective foldback precursors. We propose a model for miRNA evolution that suggests a mechanism for de novo generation of new miRNA genes with unique target specificities.

Arabidopsis↗
PubMed1999

Different gene expression of MDM2, GAGE-1, -2 and FHIT in hepatocellular carcinoma and focal nodular hyperplasia.

Overexpression and/or mutations of oncogenes, tumour suppressor genes and tumour rejection genes have been observed in several human malignancies. Their analyses might be of diagnostic importance. Therefore, malignant hepatocytes derived from hepatocellular carcinoma (HCC) tissue as well as non-malignant hepatocytes derived from focal nodular hyperplasia (FNH) were studied. Samples containing normal human hepatocytes (HC) served as controls. Cellular material was obtained by fine-needle aspiration biopsy guided by ultrasound. Cells were analysed for expression and mutation of the oncogene MDM2, the genes GAGE-1, -2 coding for tumour-associated antigens and the candidate tumour suppressor gene FHIT. Different patterns of non-mutant FHIT transcripts including precise deletion of exons were found in 7/10 HCC, 2/10 FNH and 2/10 HC. However, expression of non-mutant GAGE-1, -2 RNA was demonstrated exclusively in 6/10 HCC samples. Further genetic features specific of HCC were point mutations in a zinc-finger motif of MDM2 (3/10 HCC samples). Neither GAGE-1, -2 expression nor MDM2 mutations were observed in the FNH samples, or in normal hepatocytes. Our findings suggest that occurrence of variable FHIT transcripts is not restricted to hepatic malignant tumours. In contrast, MDM2 mutations and GAGE-1, -2 expression were associated with HCC specimens. Therefore, the RT-PCR assays for GAGE-1, -2 and MDM2 might be useful adjuncts in cytodiagnosis of liver neoplasms.

Acid Anhydride Hydrolases↗
PubMed2004

Discordant response to HAART: reduction of viremia and replicative capacity of HIV strains in patients after genotype guided change of therapy.

The aim of this study was to evaluate outcome after a genotype guided change of therapy in 18 patients failing HAART. Patients were divided into two groups according to the response to therapy: immune responders (12 patients with immune recovery defined as having more than 100 CD4 cells compared to baseline value), and 6 failing patients (without immune recovery). At month 12 after genotype change of therapy a significant difference in the decrease of HIV-RNA viral load between the two groups of patients was detected (mean -1.95 and +0.04 log HIV-RNA copies/ml, p=0.04). One year after the change of therapy, all but one patients experienced a decrease in the replication capacity of HIV strains. Particularly, the HIV replication capacity of HIV strains decreased from 52% (range 14-98%) to 15.2% (range 0.1-74.5%). The HIV strains of patients failing HAART showed a progressive impaired replication capacity. In patients failing HAART the impaired replication capacity of HIV strains could justify the persistence of an immune recovery.

Adult↗
PubMed2026

mRNA therapy: A novel approach for retinal neurodegenerative diseases.

Retinal neurodegeneration remains a major cause of irreversible vision loss, yet current therapeutic options are limited in effectiveness. Although gene therapies have shown clinical potential, the overexpression platforms they rely on, such as adeno-associated virus DNA, are constrained by safety concerns, limited efficacy, and cargo size restrictions. In contrast, mRNA therapy has gained recognition as a compelling alternative, enabling rapid and efficient protein expression without the risk of genomic integration. This review synthesizes recent advances in mRNA engineering, delivery systems, and administration routes for retinal applications, and highlight strategies to enhance targeting, penetration, and controlled release through interdisciplinary collaboration between ophthalmology and bioengineering. In recent years, engineered mRNA formats, including chemically modified linear, circular, and self-amplifying RNA, can achieve higher translation efficiency within a tunable expression window. The transient nature and relatively low immunogenicity of in vitro transcribed mRNA support repeat dosing without insertional mutagenesis. Advances in nanocarriers, particularly lipid nanoparticles, have enabled preferential delivery to retinal neurons, Müller glia, and pigment epithelium via intraocular administration, while improving mRNA stability and transfection efficiency. In preclinical studies, mRNA has been widely used to deliver gene-editing tools, transcription factors, and supplementary functional proteins. In disease models such as optic nerve crush and laser-induced choroidal neovascularization, mRNA-based therapies enhance neuroprotection and suppress pathological angiogenesis in the injured retina, with favorable ocular safety profiles. However, it remains largely unexplored how the intrinsic advantages of mRNA therapy can be leveraged to develop tailored strategies for complex retinal disorders. Consistent with this gap, mRNA platforms have not yet been widely incorporated into retinal research or clinical practice. In parallel, clinical translation also lags: despite encouraging outcomes of lipid nanoparticle-mRNA formulations in preclinical models, no candidates have progressed into retinal clinical trials. This review draws on the complex pathology and therapeutic logic of retinal neurodegeneration. It proposes that mRNA therapy enables multitarget, repeatable, stage-specific interventions that align with the dynamic evolution of diseases and the requirements of combination therapy in retinal diseases. It may be used to support neuroprotection, axon regeneration, and neurovascular regulation. By integrating data across experimental models and modalities, this review outlines representative cases and experimental paradigms to guide rational trial design and carrier selection. Taken together, technical progress and evolving application strategies position mRNA therapy as a compelling therapeutic avenue for retinal neurodegeneration.

administration↗
PubMed1997

Biochemical and molecular biological characterization of CAC2, the Arabidopsis thaliana gene coding for the biotin carboxylase subunit of the plastidic acetyl-coenzyme A carboxylase.

The biotin carboxylase subunit of the heteromeric chloroplastic acetyl-coenzyme A carboxylase (ACCase) of Arabidopsis thaliana is coded by a single gene (CAC2), which is interrupted by 15 introns. The cDNA encodes a deduced protein of 537 amino acids with an apparent N-terminal chloroplast-targeting transit peptide. Antibodies generated to a glutathione S-transferase-CAC2 fusion protein react solely with a 51-kD polypeptide of Arabidopsis; these antibodies also inhibit ACCase activity in extracts of Arabidopsis. The entire CAC2 cDNA sequence was expressed in Escherichia coli and the resulting recombinant biotin carboxylase was enzymatically active in carboxylating free biotin. The catalytic properties of the recombinant biotin carboxylase indicate that the activity of the heteromeric ACCase may be regulated by light-/dark-induced changes in stromal pH. The CAC2 gene is maximally expressed in organs and tissues that are actively synthesizing fatty acids for membrane lipids or oil deposition. The observed expression pattern of CAC2 mirrors that previously reported for the CAC1 gene (J.-K. Choi, F. Yu, E.S. Wurtele, B.J. Nikolau [1995] Plant Physiol 109: 619-625; J. Ke, J.-K. Choi, M. Smith, H.T. Horner, B.J. Nikolau, E.S. Wurtele [1997] Plant Physiol 113: 357-365), which codes for the biotin carboxyl carrier subunit of the heteromeric ACCase. This coordination is probably partially established by coordinate transcription of the two genes. This hypothesis is consistent with the finding that the CAC2 and CAC1 gene promoters share a common set of sequence motifs that may be important in guiding the transcription of these genes.

Acetyl-CoA Carboxylase↗
PubMed2000

Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis.

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses. html) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5' terminus of the user-specified primer to the 3' terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms.

Bacteria↗
PubMed1996

Specimen collection and storage for diagnostic molecular pathology investigation.

The success of the newest discipline in the diagnostic clinical pathology laboratory, molecular pathology, is dependent on proper collection and storage of both the original and processed (nucleic acid) specimen. This issue will grow in importance as test volumes increase in the diagnostic molecular pathology laboratory. This review is a distillation of a literature review by the Patient Preparation and Specimen Handling Committee of the College of American Pathologists. It describes specific collection, storage, and anticoagulant or preservative requirements based on the diagnostic molecular technique and/or specimen type used for analysis. This review serves as a guide for clinical laboratories interested in appropriate collection and storage of specimens to be used in nucleic acid-based analysis.

DNA↗
PubMed2003

On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.

Bacterial Proteins↗
PubMed2001

A single metal ion plays structural and chemical roles in an aminoacyl-transferase ribozyme.

Catalytically active RNA molecules rely on metal ions for structural and/or catalytic functions. Our in vitro selected aminoacyl-transferase ribozyme is no exception, as it employs a single fully hydrated Mg2+ ion for catalysis [Suga, H., et al. (1998) Biochemistry 37, 10118-10125]. Here we report the essential catalytic residues of the ribozyme and their spatial arrangement in the relation to the metal binding site. Evidence obtained using a combination of Pb2+ and Tb3+ hydrolytic cleavage assays on wild type and mutant ribozymes revealed a cooperative metal binding site that consists of the tandem G:U wobble pairs in P1 and consecutive G:U and U:A pairs in P3. The formation of this concerted Mg2+ binding site positions the P1 and P3 helices in a parallel manner, placing the L3 tetraloop in close proximity to the internal guide sequence (IGS, substrate binding site), which is adjacent to P1. Certain monovalent metal ions inhibit catalysis at low concentrations but support catalysis at high concentrations. These analyses imply that the Mg2+ ion plays both structural and chemical roles and that it brings about the significant rate acceleration in aminoacyl-transfer in concert with the L3-IGS long-range interaction.

Aminoacyltransferases↗
PubMed2025

An Integrated Clinical Genomic and Transcriptomic Subgrouping of Central Chondrosarcoma.

Central conventional chondrosarcoma, a malignant cartilage-producing bone tumor, is the second most common bone sarcoma. Chondrosarcomas are histologically graded, which is so far the best predictor of survival. Early mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are frequent, leading to the production of the oncometabolite D-2-hydroxyglutarate, which affects DNA methylation, resulting in a preferred chondrogenic differentiation over osteogenic differentiation of mesenchymal stem cells, which are currently considered the precursor cells of chondrosarcoma. DNA methylation profiling has previously revealed distinct profiles between IDH-mutant and IDH-wild-type chondrosarcomas, but the presence of further DNA methylation subgroups indicates that classification based solely on IDH status is too simplistic. In this study, we aim to identify biological subgroups in a total of 116 chondrosarcomas by integrating clinical data, IDH mutation status, gene expression, and genome-wide loss of heterozygosity (LOH). Clinical associations were observed between several factors, including sex and histological grade, as well as tumor site and IDH mutation status. RNA sequencing and genome-wide LOH confirmed the distinction between IDH-wild-type and IDH-mutant chondrosarcomas, where the number of chromosome arms affected by LOH was significantly higher in IDH-wild-type tumors than in IDH-mutant tumors. However, no clear subgroups emerged within each IDH group. Further clustering on RNA expression of differentiation markers identified subgroups characterized by chondrogenic, osteogenic, resting chondrocyte, or dedifferentiated profiles. These different subgroups showed a specific clinical presentation and suggest different precursor cells. Instead of a simple dichotomy between IDH-mutant and IDH-wild-type, our integrated approach highlights interconnected clinical, genomic, and transcriptomic patterns that offer a more nuanced view of chondrosarcoma biology and might potentially guide treatment stratification.

Humans↗
PubMed2006

Dual targeting of GyrB and ParE by a novel aminobenzimidazole class of antibacterial compounds.

A structure-guided drug design approach was used to optimize a novel series of aminobenzimidazoles that inhibit the essential ATPase activities of bacterial DNA gyrase and topoisomerase IV and that show potent activities against a variety of bacterial pathogens. Two such compounds, VRT-125853 and VRT-752586, were characterized for their target specificities and preferences in bacteria. In metabolite incorporation assays, VRT-125853 inhibited both DNA and RNA synthesis but had little effect on protein synthesis. Both compounds inhibited the maintenance of negative supercoils in plasmid DNA in Escherichia coli at the MIC. Sequencing of DNA corresponding to the GyrB and ParE ATP-binding regions in VRT-125853- and VRT-752586-resistant mutants revealed that their primary target in Staphylococcus aureus and Haemophilus influenzae was GyrB, whereas in Streptococcus pneumoniae it was ParE. In Enterococcus faecalis, the primary target of VRT-125853 was ParE, whereas for VRT-752586 it was GyrB. DNA transformation experiments with H. influenzae and S. aureus proved that the mutations observed in gyrB resulted in decreased susceptibilities to both compounds. Novobiocin resistance-conferring mutations in S. aureus, H. influenzae, and S. pneumoniae were found in gyrB, and these mutants showed little or no cross-resistance to VRT-125853 or VRT-752586 and vice versa. Furthermore, gyrB and parE double mutations increased the MICs of VRT-125853 and VRT-752586 significantly, providing evidence of dual targeting. Spontaneous frequencies of resistance to VRT-752586 were below detectable levels (<5.2x10(-10)) for wild-type E. faecalis but were significantly elevated for strains containing single and double target-based mutations, demonstrating that dual targeting confers low levels of resistance emergence and the maintenance of susceptibility in vitro.

Anti-Bacterial Agents↗
PubMed2006

Genetic differentiation of appendiceal tumor malignancy: a guide for the perplexed.

OBJECTIVE: To use differential gene expression of candidate markers to discriminate benign appendiceal carcinoids (APCs) from malignant and mixed cell APCs. SUMMARY BACKGROUND DATA: Controversy exists in regard to the appropriate surgical management of APCs since it is sometimes difficult to predict tumor behavior using traditional pathologic criteria. We have identified 5 differentially expressed genes (a mitosis-regulatory gene NAP1L1, an adhesin MAGE-D2, an estrogen-antagonist, the metastasis marker MTA1, the apoptotic marker NALP, and chromogranin A) that define gut neuroendocrine cell behavior. METHODS: Total RNA was isolated using TRIzol reagent from 42 appendiceal samples, including appendiceal carcinoids identified at exploration for appendicitis (no evidence of metastasis; n = 16), appendicitis specimens (n = 11), malignant appendiceal tumors (> 1.5 cm, evidence of metastatic invasion; n = 7), and mixed (goblet) cell appendiceal adenocarcinoids (n = 3), normal appendiceal tissue (n = 5), and 5 colorectal cancers. Gene expression (CgA, NAP1L1, MAGE-D2, MTA1, and NALP1) was examined by Q-RT PCR (Applied Biosystems) and quantified against GAPDH. RESULTS: CgA message was elevated (> 1000-fold, P < 0.05) in all tumor types. NAP1L1 was elevated (> 10-fold, P < 0.03) in both malignant and goblet cell adenocarcinoids compared with normal and incidental lesions (P < 0.006). MAGE-D2 and MTA1 message were significantly elevated (> 10-fold, P < 0.01) in the malignant and goblet cell adenocarcinoid tumors but not in the appendicitis-associated carcinoids or normal mucosa. The apoptotic marker, NALP1, was overexpressed (> 50-fold, P < 0.05) in the appendicitis-associated and malignant appendiceal carcinoids but was significantly decreased (> 10-fold, P < 0.05) in the goblet cell adenocarcinoids. Elevated CgA transcript and protein levels indicative of a carcinoid tumor were identified in one acute appendicitis sample with no histologic evidence of a tumor. CONCLUSIONS: These data demonstrate that malignant APCs and goblet cell adenocarcinoids have elevated expression of NAP1L1, MAGE-D2, and MTA1 compared with appendiceal carcinoids identified at surgery for appendicitis. This and the differences in NALP1 gene expression (decreased in goblet cell adenocarcinoids) provide a series of molecular signatures that differentiate carcinoids of the appendix. CgA identified all appendiceal tumors as well as covert lesions, which may be more prevalent than previously recognized. The molecular delineation of malignant appendiceal tumor potential provides a scientific basis to define the appropriate surgical management as opposed to morphologic assessment alone.

Adaptor Proteins, Signal Transducing↗