Impact of monocytic cells on recovery of uncultivable bacteria from atherosclerotic lesions.

OBJECTIVE: Epidemiological evidence suggests that infections may contribute to atherogenesis. However, with the exception of Chlamydophila pneumoniae, cultivable bacteria have not been recovered from atherosclerotic lesions. Therefore, we aimed at developing an approach to recover uncultivable bacteria from atherectomy tissues. METHODS: We cultured homogenates from atherectomy specimens from seven nonseptic patients undergoing surgery for arterial obstruction either alone or together with THP-1 monocyte-like cells. We performed 16S rDNA analysis, biochemical tests, random amplification of polymorphic DNA PCR analysis, quantitative polymerase chain reaction (qPCR) and immunohistofluorescence to identify the cultivated bacteria. Wilcoxon signed-rank tests were used to determine whether THP-1 treatment yielded a higher number of isolates than did the untreated controls. RESULTS: We recovered more bacteria from cocultures of atherectomy specimens with THP-1 cells than atherectomy specimens cultured alone. On average, tissue homogenates incubated with THP-1 cells versus control yielded 124 vs. 22 colony-forming units, a median of 140 vs. 7, respectively (P = 0.02). We recovered 872 isolates of limited number of species, including Propionibacterium acnes, Staphylococcus epidermidis and Streptococcus infantis and the fastidious anaerobe Porphyromonas gingivalis, and confirmed its presence in tissue using double immunofluorescence imaging. qPCR demonstrated the presence of ≥3.5 × 10(3) P. gingivalis genomes per gram of atheromatous tissue. CONCLUSIONS: These results indicate that viable previously uncultivable bacterial species are present within atheromas. Our results suggest revisiting the hypothesis that infections may have a causative role in atherosclerotic inflammation and have implications for research regarding novel diagnostics and treatments for cardiovascular disease.

Classification of partial seizure symptoms in genetic studies of the epilepsies.

OBJECTIVES: To develop standardized definitions for classification of partial seizure symptoms for use in genetic research on the epilepsies, and evaluate inter-rater reliability of classifications based on these definitions. METHODS: The authors developed the Partial Seizure Symptom Definitions (PSSD), which include standardized definitions of 41 partial seizure symptoms within the sensory, autonomic, aphasic, psychic, and motor categories. Based on these definitions, two epileptologists independently classified partial seizures in 75 individuals from 34 families selected because one person had ictal auditory symptoms or aphasia. The data used for classification consisted of standardized diagnostic interviews with subjects and family informants, and medical records obtained from treating neurologists. Agreement was assessed by kappa. RESULTS: Agreement between the two neurologists using the PSSD was "substantial" or "almost perfect" for most symptom categories. CONCLUSIONS: Use of standardized definitions for classification of partial seizure symptoms such as those in the Partial Seizure Symptom Definitions should improve reliability and accuracy in future genetic studies of the epilepsies.

LGI1 mutations in autosomal dominant partial epilepsy with auditory features.

OBJECTIVE: S: Mutations in LGI1 cause autosomal dominant partial epilepsy with auditory features (ADPEAF), a form of familial temporal lobe epilepsy with auditory ictal manifestations. The authors aimed to determine what proportion of ADPEAF families carries a mutation, to estimate the penetrance of identified mutations, and to identify clinical features that distinguish families with and without mutations. METHODS: The authors sequenced LGI1 in 10 newly described ADPEAF families and analyzed clinical features in these families and others with mutations reported previously. RESULTS: Three of the families had missense mutations in LGI1 (C42R, I298T, and A110D). Penetrance was 54% in eight families with LGI1 mutations the authors have identified so far (five reported previously and three reported here). Excluding the original linkage family, the authors have found mutations in 50% (7/14) of tested families. Families with and without mutations had similar clinical features, but those with mutations contained significantly more subjects with auditory symptoms and significantly fewer with autonomic symptoms. In families with mutations, the most common auditory symptom type was simple, unformed sounds (e.g., buzzing and ringing). In two of the newly identified families with mutations, some subjects with mutations had idiopathic generalized epilepsies. CONCLUSIONS: LGI1 mutations are a common cause of autosomal dominant partial epilepsy with auditory features. Current data do not reveal a clinical feature that clearly predicts which families with autosomal dominant partial epilepsy with auditory features have a mutation. Some families with LGI1 mutations contain individuals with idiopathic generalized epilepsies. This could result from either an effect of LGI1 on risk for generalized epilepsy or an effect of co-occurring idiopathic generalized epilepsy-specific genes in these families.

Nucleotide sequence, transcription map, and mutation analysis of the 13q14 chromosomal region deleted in B-cell chronic lymphocytic leukemia.

Deletions of the 13q14 chromosome region are associated with B-cell chronic lymphocytic leukemia (B-CLL) and several other types of cancer, suggesting the presence of a tumor suppressor gene. In previous studies the minimal region of deletion (MDR) was mapped to a less than 300-kilobase (kb) interval bordered by the markers 173a12-82 and 138G4/1.3R. For the identification of the putative tumor suppressor gene, the entire MDR (approximately 347 kb) has been sequenced, and transcribed regions have been identified by exon trapping, EST-based full-length complementary DNA cloning, database homology searches, and computer-assisted gene prediction analyses. The MDR contains 2 pseudogenes and 3 transcribed genes: CAR, encoding a putative RING-finger containing protein; 1B4/Leu2, generating noncoding transcripts; and EST70/Leu1, probably representing another noncoding gene (longest open reading frame of 78 codons). These genes have been sequenced in 20 B-CLL cases with 13q14 hemizygous deletion, and no mutations were found. Moreover, no somatic variants were found in the entire MDR analyzed for nucleotide substitutions by a combination of direct sequencing and fluorescence-assisted mismatch analysis in 5 B-CLL cases displaying 13q14-monoallelic deletion. The nondeleted allele of the CAR and EST70/Leu1 genes was expressed in B-CLL specimens, including those with monoallelic loss, whereas no expression of 1B4/Leu2 was detectable in B-CLL, regardless of the 13q14 status. These results indicate that allelic loss and mutation of a gene within the MDR is an unlikely pathogenetic mechanism for B-CLL. However, haplo-insufficiency of one of the identified genes may contribute to tumorigenesis. (Blood. 2001;97:2098-2104)

Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene.

Familial primary pulmonary hypertension is a rare autosomal dominant disorder that has reduced penetrance and that has been mapped to a 3-cM region on chromosome 2q33 (locus PPH1). The phenotype is characterized by monoclonal plexiform lesions of proliferating endothelial cells in pulmonary arterioles. These lesions lead to elevated pulmonary-artery pressures, right-ventricular failure, and death. Although primary pulmonary hypertension is rare, cases secondary to known etiologies are more common and include those associated with the appetite-suppressant drugs, including phentermine-fenfluramine. We genotyped 35 multiplex families with the disorder, using 27 microsatellite markers; we constructed disease haplotypes; and we looked for evidence of haplotype sharing across families, using the program TRANSMIT. Suggestive evidence of sharing was observed with markers GGAA19e07 and D2S307, and three nearby candidate genes were examined by denaturing high-performance liquid chromatography on individuals from 19 families. One of these genes (BMPR2), which encodes bone morphogenetic protein receptor type II, was found to contain five mutations that predict premature termination of the protein product and two missense mutations. These mutations were not observed in 196 control chromosomes. These findings indicate that the bone morphogenetic protein-signaling pathway is defective in patients with primary pulmonary hypertension and may implicate the pathway in the nonfamilial forms of the disease.

A knowledge model for analysis and simulation of regulatory networks.

MOTIVATION: In order to aid in hypothesis-driven experimental gene discovery, we are designing a computer application for the automatic retrieval of signal transduction data from electronic versions of scientific publications using natural language processing (NLP) techniques, as well as for visualizing and editing representations of regulatory systems. These systems describe both signal transduction and biochemical pathways within complex multicellular organisms, yeast, and bacteria. This computer application in turn requires the development of a domain-specific ontology, or knowledge model. RESULTS: We introduce an ontological model for the representation of biological knowledge related to regulatory networks in vertebrates. We outline a taxonomy of the concepts, define their 'whole-to-part' relationships, describe the properties of major concepts, and outline a set of the most important axioms. The ontology is partially realized in a computer system designed to aid researchers in biology and medicine in visualizing and editing a representation of a signal transduction system.

Cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21.

The beclin 1 (BECN1) gene encodes a 60-kDa coiled-coil protein that interacts with the prototypic apoptosis inhibitor Bcl-2. Previous studies indicate that beclin 1 maps to a region approximately 150 kb centromeric to BRCA1 on chromosome 17q21 that is commonly deleted in breast, ovarian, and prostate cancer. The complete cDNA sequence of beclin 1 encodes a 2098-bp transcript, with a 120-bp 5' UTR, 1353-bp coding region, and 625-bp 3' UTR. Hybridization screening of a human genomic PAC library identified PAC 452O8, which contains the complete beclin 1 gene. Determination of the exon-intron structure of beclin 1 reveals 12 exons, ranging from 61 to 794 bp, which extend over 12 kb of the human genome. FISH analysis of human breast carcinoma cell lines using PAC 452O8 as probe identified allelic beclin 1 deletions in 9 of 22 cell lines. Sequencing of genomic DNA from 10 of these cell lines revealed no mutations in coding regions or splice junctions. Additionally, Northern blot analysis of 11 cell lines did not identify any abnormalities in beclin 1 transcripts. These results indicate that human breast carcinoma cell lines frequently contain allelic deletions of beclin 1, but not beclin 1 coding mutations.

Molecular hybridization probes prepared with 4-aminooxybutylamine.

A versatile method is described for preparing nonradioactive DNA probes for molecular hybridization. The method is based on the transamination reaction of double-stranded DNA with 4-aminooxybutylamine (ABA). To optimize the procedure for obtaining stable and sensitive hybridization probes, time of modification, pH, and reaction temperature were varied. The optimal reaction conditions allowed the preparation of nonradioactive-labeled DNA probes that met demands for optimal length, modification degree, stability, and sensitivity. The use of 4-aminooxybutylamine as a bifunctional reagent for DNA modification allowed the possibility of choosing an appropriate reporter group: biotin or one of the fluorochromes. For probes carrying biotin, a high specificity and high sensitivity detection limit of 1 pg of target DNA were demonstrated. In addition, the applicability of probes carrying fluorochromes for multicolor direct fluorescence in situ hybridization was described.

Tools for visualization and integration of intermediate sequencing results in large disease gene discovery projects.

We describe two Java applets which are useful for insightful presentation of intermediate experimental data in gene discovery projects involving large scale sequencing. One of these applets provides a physical map of a genomic region and provides easy access to the second applet, which furnishes a detailed map of sequence contigs associated with clones on the physical map. In particular, the second applet displays all the known information about each contig, including the presence of exons, database homology 'hits', repetitive elements and other features; the graphics are linked to other World Wide Web pages, providing detailed information on each feature. These applets should be useful to other research groups working on large sequencing projects.

High-resolution YAC-cosmid-STS map of human chromosome 13.

We have assembled a high-resolution physical map of human chromosome 13 DNA (approximately 114 Mb) from hybridization, PCR, and FISH mapping data using a specifically designed set of computer programs. Although the mapping of 13p is limited, 13q (approximately 98 Mb) is covered by an almost continuous contig of 736 YACs aligned to 597 contigs of cosmids. Of a total of 10,789 cosmids initially selected from a chromosome 13-specific cosmid library (16,896 colonies) using inter-Alu PCR probes from the YACs and probes for markers mapped to chromosome 13, 511 were assembled in contigs that were established from cross-hybridization relationships between the cosmids. The 13q YAC-cosmid map was annotated with 655 sequence tagged sites (STSs) with an average spacing of 1 STS per 150 kb. This set of STSs, each identified by a D number and cytogenetic location, includes database markers (198), expressed sequence tags (93), and STSs generated by sequencing of the ends of cosmid inserts (364). Additional annotation has been provided by positioning 197 cosmids mapped by FISH on 13q. The final (comprehensive) map, a list of STS primers, and raw data used in map assembly are available at our Web site (genome1.ccc.columbia.edu/ approximately genome/) and can serve as a resource to facilitate accurate localization of additional markers, provide substrates for sequencing, and assist in the discovery of chromosome 13 genes associated with hereditary diseases.

A single gene for human TRAF-3 at chromosome 14q32.3 encodes a variety of mRNA species by alternative polyadenylation, mRNA splicing and transcription initiation.

Human TRAF-3 is a signaling molecule that interacts with the cytoplasmic tails of CD40 and other TNF-receptor family members. TRAF-3 mRNA is expressed as two major classes of approximately 2 and 8 kb and a number of TRAF-3 encoding cDNA clones differ in discrete gene segments. Because this variety of mRNA species could result from mRNA processing events and/or multiple genes, the structure and localization of TRAF-3 encoding gene elements were determined. FISH and radiation hybrid mapping demonstrated that TRAF-3 is located at chromosome 14q32.3, approximately 1 Mb centromeric to the Ig heavy chain gene complex. Physical mapping of four overlapping genomic PAC clones established that TRAF-3 transcripts are encoded by a single gene, comprised of 13 exons and spanning 130 kb. Alternative polyadenylation in the mRNA segment encoded by exon 12 accounts for the difference between the 2 kb and the 8 kb classes of transcripts. Alternative mRNA splicing in the coding region (encoded by exons 3-12) generates transcripts which delete exons 8 (75 nt), 7+8 (156 nt) or 8+9 (168 nt) and that encode distinct protein isoforms (delta25, delta52 and delta56 aa, respectively). Alternative splicing of exon 2 (139 nt) and alternative transcriptional initiation result in mRNA species with distinct 5'UTRs. Together, these data indicate that a single TRAF-3 gene encodes a variety of mRNA species by a combination of alternative polyadenylation, alternative mRNA splicing and/or alternative initiation.

TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells.

A novel member of the tumor necrosis factor (TNF) cytokine family, designated TRANCE, was cloned during a search for apoptosis-regulatory genes using a somatic cell genetic approach in T cell hybridomas. The TRANCE gene encodes a type II membrane protein of 316 amino acids with a predicted molecular mass of 35 kDa. Its extracellular domain is most closely related to TRAIL, FasL, and TNF. TRANCE is an immediate early gene up-regulated by TCR stimulation and is controlled by calcineurin-regulated transcription factors. TRANCE is most highly expressed in thymus and lymph nodes but not in nonlymphoid tissues and is abundantly expressed in T cells but not in B cells. Cross-hybridization of the mouse cDNA to a human thymus library yielded the human homolog, which encodes a protein 83% identical to the mouse ectodomain. Human TRANCE was mapped to chromosome 13q14 while mouse TRANCE was located to the portion of mouse chromosome 14 syntenic with human chromosome 13q14. A recombinant soluble form of TRANCE composed of the entire ectodomain induced c-Jun N-terminal kinase (JNK) activation in T cells but not in splenic B cells or in bone marrow-derived dendritic cells. These results suggest a role for this TNF-related ligand in the regulation of the T cell-dependent immune response.

Cloning and gene mapping of the chromosome 13q14 region deleted in chronic lymphocytic leukemia.

Frequent deletions and loss of heterozygosity in a segment of chromosome 13 (13q14) in cases of B-cell chronic lymphocytic leukemia (CLL) have suggested that this malignancy is caused by inactivation of an unknown tumor suppressor gene located in this region. Toward the identification of the putative CLL tumor suppressor, we have constructed a high-resolution physical map of YAC, PAC, and cosmid contigs covering 600 kb of the 13q14 genomic region. In addition to densely positioned genetic markers and STSs, this map was further annotated by localization of 32 transcribed sequences (ESTs) using a combination of exon trapping, direct cDNA selection, sample sequencing of cosmids and PACs, and homology searches. On the basis of these mapping data, allelic loss analyses at 13q14 using CLL tumor samples allowed narrowing of the genomic segment encompassing the putative CLL gene to <300 kb. Twenty-three ESTs located within this minimally deleted region are candidate exons for the CLL-associated tumor suppressor gene.

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

[Cloning universal probes for detecting mycoplasma contamination of cell cultures]. / Kolnirovanie universal'nykh zondov dlia vyiavleniia mikoplazmennogo zarazheniia kletochnykh kul'tur.

To obtain the universal polynucleotide hybridization probes for testing mycoplasmal contaminations in cell cultures, we have cloned several DNA fragments from the srRNA gene of Acholeplasma laidlawii. Before cloning, in order to exclude cross-hybridization of these probes with eukaryotic rRNA, the thermodynamic parameters of duplex formation between DNA complementary to mycoplasmal rRNA and eukaryotic rRNA had been studied. Using a set of computer methods, the region which forms weak heteroduplexes with eukaryotic srRNA was revealed. This region occupies positions 250 to 550 position of the mycoplasmal srRNA. Three different DNA fragments which include the region were generated in PCR, cloned in pUC18, and their hybridization characteristics were evaluated. In appropriate hybridization conditions the probes hybridize with all mycoplasmal RNAs studied without cross-hybridization with eukaryotic ribosomal RNA and DNA, and allow one to detect virtually any mycoplasmas (or any prokaryote) in cell cultures. Blot-hybridization of universal probes with mycoplasmal DNA digested by BsuRI allows one to identify the different species of mycoplasmas.

[The isolation of the bovine leukemia provirus by the polymerase chain reaction with subsequent nonradioactive blot hybridization]. / Vyiavlenie provirusa leikoza krupnogo rogatogo skota metodom polimeraznoi tsepnoi reaktsii s posleduiushchei neradioaktivnoi blot-gibridizatsiei.

A multiprobe scheme for detecting provirus of bovine leukemia virus (BLV) in the peripheral blood of infected animals was developed. According to the scheme, a fragment of BLV X-gene was amplified by polymerase chain reaction and detected by blot hybridization with the biotinylated oligonucleotide probe complementary to the inner part of the generated fragment. 600 copies of provirus may be specifically detected in a sample containing 150,000 cells. In some cases, BLV was detected in blood samples negative if tested by the commercial double immunodiffusion test.

[Immobilization of DNA on microporous membranes using UV-irradiation]. / Immobilizatsiia DNK na mikroporistykh membranakh s pomoshch'iu UF- oblucheniia.

Various techniques of DNA immobilization onto nitrocellulose and nylon microporous membranes have been compared. Despite a strong primary adsorption of DNA onto these membranes during blotting procedures, poor retention of the target DNA and low hybridization signals are obtained after hybridization and washings. Covalent cross-linking of DNA upon UV irradiation leads to a quantitative immobilization of target DNA. Quantum yield of DNA photoimmobilization estimated for a single base in DNA is about 10(-4). UV irradiation dose sufficient for immobilization of DNA fragment of a known length can be calculated by the formula Ilc = (22.3 +/- 4.8) c/l, where l is the DNA fragment length (in base pairs), c is the DNA part (%) to be immobilized. The UV irradiation dose about 0.6-0.8 kJ/m2 is optimal for most hybridization experiments. Doses higher than 0.8-1 kJ/m2 may cause a loss in the hybridization efficiency. Under optimal immobilization conditions, hybridization signals increasing five-fold for nitrocellulose membranes and fifty-fold for uncharged nylon membranes as compared with baking these membranes in vacuum.

[DNA carrying aliphatic amino groups and the use of its fluorescent derivative as a probe in molecular hybridization]. / Izuchenie DNK, nesushchei alifaticheskie aminogruppy, i ispol'zovanie ee fluorestsentnogo proizvodnogo v kachestve zonda pri molekuliarnoi gibridizatsii.

A two-step method of labelling DNA through aliphatic amino groups with various reporter molecules has been developed. The method is based on reaction of cytosine-residues of polynucleotide chain with 4-aminooxybutylamine; the latter's synthesis is described. DNA modified on this way with fluorescein isothiocyanate functions a probe in molecular hybridisation. The sensitivity of this method for fluorochrome-labelled DNA probes in the dot hybridisation procedure is about 200 pg per mm2.