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PubMed2002

The Babesia bovis merozoite surface antigen 2 locus contains four tandemly arranged and expressed genes encoding immunologically distinct proteins.

Members of the variable merozoite surface antigen (vmsa) gene family of Babesia bovis encode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containing msa-2, a member of the vmsa family, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies of msa-2-related genes were found in the locus. The four genes, designated msa-2a(1) (which corresponds to the originally described msa-2 gene), msa-2a(2), msa-2b, and msa-2c, were shown to be transcribed and expressed and encode proteins with open reading frames ranging in size from 266 (MSA-2c) to 317 (MSA-2a(1)) amino acids. MSA-2a(1) and -2a(2) are the most closely related of the four proteins (90% identity), differing by (i) the number of 24-amino-acid repeats that comprise a surface-exposed B-cell epitope and (ii) the presence of a 32-amino-acid area of recombination between MSA-2a(2) and -2b. In contrast, msa-2c is most closely related to the previously described babr 0.8 gene in Australia strains of B. bovis. Comparison of MSA-2 proteins in the Argentina R1A strain of B. bovis with the Mexico Mo7 clone revealed a relatively high degree of conservation (83.6, 69.4, 79.1, and 88.7% amino acid identity for MSA-2a(1), -2a(2), -2b, and -2c, respectively), in contrast to the extensive MSA-1 sequence variation (52% identity) between the same two strains. Postinfection bovine immune serum contains antibodies that bound to each of the recombinant MSA-2 proteins. Blocking assays demonstrated the presence of unique B-cell epitopes in MSA-2a(1), -2b, and -2c. The results support the evolution of the msa-2 locus through at least two gene duplications, with selection for multiple related but antigenically distinct merozoite surface proteins.

Amino Acid Sequence↗

PubMed1997

Copia-like retrotransposons in rice: sequence heterogeneity, species distribution and chromosomal locations.

Degenerated oligonucleotide primers were used to amplify, clone, and analyze sequence heterogeneity and chromosomal distribution of 23 PCR fragments corresponding to the reverse transcriptase domain of copia-like retrotransposons in rice. Of the 23 fragments 22 could be aligned by their deduced amino acid sequences and were divided into 6 groups according to the phylogenetic and Southern blot analyses. Amino acid sequence differences among the 22 aligned fragments ranged from 1 to 64%. Southern blot analysis of 10 rice accessions including indica, japonica and common wild rice, using these 23 fragments as probes, showed that copia-like retrotransposons were present in moderate to high copy numbers in all the rice genome although the exact copy number cannot be determined. The major difference revealed by southern analysis is a differentiation between the four indica varieties as one group and the four japonica varieties and the two wild rice accessions as another group. Polymorphisms were also detected among the indica and japonica varieties by major bands and repeatable minor bands. Five hybridization bands were mapped to chromosomes 3, 4, 8, and 9, respectively. All the five bands were inherited in a dominant Mendelian fashion and were not allelic with each other, indicating that the same element did not reside on the same location in different rice accessions. No transcript of the copia-like reverse transcriptase was detected on northern blot. The results suggest that the sequence heterogeneity and distributional variability of retrotransposons may be one of contributory factors causing genetic diversity in rice.

Amino Acid Sequence↗

PubMed1996

Quantification of HIV-1 proviral DNA and analysis of genomic diversity by polymerase chain reaction and temperature gradient gel electrophoresis.

A competitive polymerase chain reaction/temperature gradient gel electrophoresis (PCR/TGGE) protocol was developed for exact quantification of HIV-1 proviral DNA copy numbers in clinical samples. An internal standard (ST) that differs from wildtype-sequences only by a single base exchange was used as a competitor in PCR. Quantification of HIV-1 target sequences was achieved by coamplification of defined copy numbers of ST with wild type target sequences, hybridization of PCR products to a strand-specifically labelled probe, separation of ST and wildtype sequences by TGGE, and determination of the ratio of wildtype and standard sequences by densitometric scanning. Effects of sample preparation, DNA extraction and white blood cell counts were minimized by the additional quantification of beta-globin sequences. With this technique, it was possible to determine precisely the number of HIV target sequences as compared to the number of beta-globin gene copies with a detection limit of two HIV-1 proviral copies. Forty-four peripheral blood mononuclear cell (PBMC) extracts from 39 HIV-1 infected patients were analyzed by PCR/TGGE. HIV-1 proviral DNA levels ranged between 2 and 24190 HIV-copies/10(6) beta-globin copies. In general, patients in the advanced stages of disease and/or with low CD4 counts had much higher proviral DNA levels than patients in early stages or with high CD4 counts. In patients from whom consecutive samples were obtained, progression of disease correlated with a greater than tenfold rise of HIV-copies/10(6) beta-globin copies. Compared to other recently published protocols for proviral DNA quantification, this experimental approach allows in addition direct demonstration of mutations within the amplified region. The competitive PCR/TGGE protocol described in this study is suitable for monitoring fluctuations of proviral DNA levels and to identify the genomic diversity of HIV target sequences simultaneously in one assay.

Base Sequence↗

PubMed1999

Occurrence and expression of glutathione-S-transferase-encoding bphK genes in Burkholderia sp. strain LB400 and other biphenyl-utilizing bacteria.

The gene bphK of Burkholderia sp. strain LB400 has previously been shown to be located within the bph locus, which specifies the degradation of biphenyl (BP) and chlorobiphenyls, and to encode a glutathione S-transferase (GST) which accepts 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The specific physiological role of this gene is not known. It is now shown that the gene is expressed in the parental organism and that GST activity is induced more than 20-fold by growth of the strain on BP relative to succinate when these compounds serve as sole carbon source. Approximately the same induction factor was observed for 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, which is encoded by the 5'-adjacent bphC gene. This suggests that the expression of bphK is coregulated with the expression of genes responsible for the catabolism of BP. A bphK probe detected only a single copy of the gene in strain LB400. A spontaneous BP- mutant of the organism neither gave a signal with the bphK probe nor showed CDNB-accepting GST activity, suggesting that this activity is solely encoded by bphK. Complementation of the mutant with a bph gene cluster devoid of bphK restored the ability to grow on BP, indicating that bphK is not essential for utilization of this carbon source. BphK activity proved to be almost unaffected by up to 100-fold differences in proton concentration or ionic strength. The enzyme showed a narrow range with respect to a variety of widely used electrophilic GST substrates, accepting only CDNB. A number of established laboratory strains as well as novel isolates able to grow on BP as sole carbon and energy source were examined for BphK activity and the presence of a bphK analogue. CDNB assays, probe hybridizations and PCR showed that several, but not all, BP degraders possess this type of GST activity and/or a closely related gene. In all bacteria showing BphK activity, this was induced by growth on BP as sole carbon source, although activity levels differed by up to 10-fold after growth on BP and by up to 60-fold after growth on succinate. This resulted in a variation of induction factors between 2 and 30. In the majority of bphK+ bacteria examined, the gene appeared to be part of LB400-like bph gene clusters. DNA sequencing revealed almost complete identity of bphK genes from five different bph gene clusters. These results suggest that bphK genes, although not essential, fulfill a strain-specific function related to the utilization of BPs by their host organisms. The usefulness of BphK as a reporter enzyme for monitoring the expression of catabolic pathways is discussed.

Biphenyl Compounds↗

PubMed2001

Antigen 43, the major phase-variable protein of the Escherichia coli outer membrane, can exist as a family of proteins encoded by multiple alleles.

agn43 encodes a major phase-variable outer-membrane protein, antigen 43 (Ag43), involved in autoaggregation of Escherichia coli cells. The gene is present in single copy on the chromosome of E. coli K-12. In contrast, Southern hybridization and gene inactivation studies demonstrate that control producer strain E. coli ML308-225 possesses duplicate copies of agn43 (agn43A and agn43B). Construction and analyses of single and double knockout mutants clearly show that both alleles are capable of expressing antigen in a phase-variable manner, with observed differences in the ON<-->OFF switch frequencies appearing to favour expression of Ag43B under conditions of normal laboratory growth. Comparative analysis of agn43A and agn43B gene sequences revealed 98% identity at the nucleotide and predicted protein levels, with differences in the protein sequence of the surface-expressed alpha(43) subunit altering the surface probability of one of the predicted epitopes. Analysis of a panel of enteropathogenic E. coli strains by Southern hybridization using agn43-specific gene probes provided strong evidence for the presence of varying numbers of agn43 alleles within clinical isolates. Taken together, the results indicate the presence of a family of distinct Ag43 proteins encoded by multiple chromosomal alleles.

Adhesins, Bacterial↗

PubMed2006

The occurrence of severe sepsis and septic shock are related to distinct patterns of cytokine gene expression.

Patient response to acute bacterial infection is highly variable. Differing outcomes in this setting may be related to variations in the immune response to an infectious insult. Using quantitative real-time polymerase chain reaction, we quantified gene expression of the tumor necrosis factor alpha(TNFalpha), interferon gamma (IFNgamma), and interleukin 10 (IL10), IL12p35, and IL4 genes in 3 patient groups. These groups consisted of an intensive care unit (ICU) cohort who presented with severe sepsis or septic shock, a group of noncritically ill ward patients with documented Gram-negative bacteremia, and a group of healthy controls. Greater interleukin 10 messenger RNA (mRNA) levels were detected in the ICU group in comparison with both the bacteremic and control groups (P < 0.0001). More TNF-alpha mRNA was detected in the ICU group when compared with the control group (P < 0.0001). However, TNF-alpha mRNA was most abundant in the bacteremic group (P = 0.0007). Lesser IFN-gamma mRNA levels were detected in the ICU group when compared with both the bacteremic and control groups (P < 0.0003). Cytokine mRNA levels were not associated with the occurrence of shock upon admission to ICU. On the seventh day of ICU stay, the presence of shock was associated with lesser IFN-gamma mRNA (P = 0.0004) and lesser TNF-alpha mRNA (P = 0.001). Survivors had greater TNF-alpha mRNA copy numbers on day 7 of ICU stay than nonsurvivors (P = 0.002). We conclude that a proinflammatory response is the appropriate response in the setting of infection and is associated with lesser requirements for inotropes and lesser mortality. Quantitative real-time polymerase chain reaction can be used to predict infection outcome in clinically relevant situations where enzyme-linked immunosorbent assay testing has proved disappointing.

Adult↗

PubMed2005

Interrater reliability: completing the methods description in medical records review studies.

In medical records review studies, information on the interrater reliability (IRR) of the data is seldom reported. This study assesses the IRR of data collected for a complex medical records review study. Elements selected for determining IRR included "demographic" data that require copying explicit information (e.g., gender, birth date), "free-text" data that require identifying and copying (e.g., chief complaints and diagnoses), and data that require abstractor judgment in determining what to record (e.g., whether heart disease was considered). Rates of agreement were assessed by the greatest number of answers (one to all n) that were the same. The IRR scores improved over time. At 1 month, the reliability for demographic data elements was very good, for free-text data elements was good, but for data elements requiring abstractor judgment was unacceptable (only 3.4 of six answers agreed, on average). All assessments after 6 months showed very good to excellent IRR. This study demonstrates that IRR can be evaluated and summarized, providing important information to the study investigators and to the consumer for assessing the reliability of the data and therefore the validity of the study results and conclusions. IRR information should be required for all large medical records studies.

Documentation↗

PubMed1990

Characterization of the repertoire of hypervariable regions in the Protein II (opa) gene family of Neisseria gonorrhoeae.

P.II outer membrane proteins of Neisseria gonorrhoeae are encoded by a family of closely related genes. Although the genes are highly conserved, major differences in sequence among them occur in two short regions, designated hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of the hypervariable regions in the P.II genes of strains FA1090. The FA1090 chromosome contained at least eleven P.II loci, having six different versions each of HV1 and HV2 among them. Southern blotting with HV-specific oligonucleotides showed that each version was present in one to three copies, and that there were nine unique combinations of HV1 and HV2 in the P.II genes. Although each of the versions of HV1 or HV2 had a unique DNA sequence, there were some similarities among them, particularly when certain ones were compared. Restriction fragments containing only the HV regions were cloned into an expression vector to demonstrate that the epitopes recognized by a set of monoclonal antibodies specific for different FA1090 P.II proteins were completely encoded by either HV1 or HV2.

Amino Acid Sequence↗

PubMed2001

Germline diversity of the expressed BALB/c VhJ558 gene family.

Although the mouse immunoglobulin heavy chain (Igh) locus contains 15 heavy chain V (Vh) gene families, at least half of the Vh gene segments are members of the VhJ558 family. This large Vh gene family represents the least characterized germline coding regions of any of the mouse antigen receptor loci and the contribution of individual VhJ558 genes to the preimmune repertoire is poorly understood. In fact, relatively few germline VhJ558 sequences have been reported for BALB/c, the foundation strain for mouse immunoglobulin genetics and the prototypic strain of the Igh(a) haplotype. Here we present a database consisting of 66 sequences estimated to represent one-half of the total number of functional BALB/c VhJ558 genes. Our results indicate that a subset of the VhJ558 genes is highly expressed in the preimmune repertoire, with just nine Vh sequences accounting for nearly 50% of the VhJ558 heavy chains expressed by splenic B cells. We show that this disparity in the expressed Vh gene repertoire is not due to the position of the Vh genes relative to the Dh cluster or to multiple germline copies of the highly expressed VhJ558 genes. Together, these data constitute the first detailed analysis of functional BALB/c VhJ558 genes, demonstrate a striking bias in the use of particular VhJ558 genes in the preimmune repertoire, and provide sufficient information to study the regulation of the Dh-distal region of the Igh-V locus at the level of individual genes.

Amino Acid Sequence↗

PubMed1997

Frequent occurrence of CYP2D6 gene duplication in Saudi Arabians.

The polymorphic cytochrome P450 2D6 (CYP2D6) causing poor, extensive or ultrarapid metabolism of several clinically important drugs exhibits pronounced interethnic variation. Ultrarapid metabolism is caused by multiple copies of active CYP2D6 genes and recently 29% of an Ethiopian population has been shown to carry duplicated or multiduplicated CYP2D6 genes, whereas the corresponding frequency in other black, Oriental and European populations investigated is 1-2%. In order to characterize the distribution of alleles with multiple CYP2D6 copies in a neighbouring population and to characterize the CYP2D locus in general among Saudi Arabians, the CYP2D6 genotype of a Saudi Arabian population was examined using restriction fragment length polymorphism (RFLP) analysis and allele-specific polymerase chain reaction (PCR) amplification. Of 101 Saudi Arabians studied, 21 subjects had an EcoRI fragment indicative of CYP2D6 gene duplication. In contrast, only two individuals were heterozygous for a deletion of the whole gene (CYP2D6*5). The allele frequency of CYP2D6*4, the most common defective allele among Caucasians, was only 3.5% in the Saudi population. Two other alleles, CYP2D6*10 and *17, common in certain populations and which cause diminished enzyme activity, were found only at low allele frequencies of 3.0% each. These findings are in agreement with earlier Saudi Arabian phenotyping studies which reported a low frequency (1-2%) of poor metabolizers for CYP2D6 probe drugs. In conclusion, the Saudi Arabian population studied exhibited very few defective alleles and a large number of subjects carried duplicated CYP2D6 genes, implying a high conservation on functional CYP2D6 genes possibly due to dietary reasons and reveal the Saudi Arabians as an unique population in comparison with others examined.

Adult↗

PubMed2000

Molecular phylogenetics of Calamus (Palmae) and related rattan genera based on 5S nrDNA spacer sequence data.

Phylogenetic relationships among the rattan palm genera Calamus, Daemonorops, Ceratolobus, Calospatha, Pogonotium, and Retispatha were investigated using DNA sequences from the nontranscribed spacer of 5S nrDNA. Moderate levels of intragenome polymorphism were identified, indicating that concerted evolution is not completely homogenizing the multiple copies of the 5S nrDNA repeat present in the nuclear genome. The existence of intragenome polymorphism did not excessively interfere with phylogeny reconstruction because, in the majority of cases, multiple clones obtained from individual species were resolved as monophyletic groups. The highly speciose genus Calamus was found to be nonmonophyletic with all five remaining genera being embedded within it. A number of major lineages within Calamus were resolved, one of which included the monotypic genus Calospatha, another included the monotypic genus Retispatha, and a third included a monophyletic group comprising Daemonorops, Ceratolobus, and Pogonotium. While the findings indicate that generic circumscriptions require revision, a nomenclatural solution was not sought at this stage because inadequate sampling and lack of support at basal nodes suggested that the topologies obtained might not be entirely reliable. Under these circumstances, name changes to such an important group would be both unhelpful and irresponsible.

Africa↗

PubMed1991

Organization of DNA sequences highly repeated in tandem in rice genomes.

Digestion of the total genomic DNA from rice Oryza sativa L. cv. C5924 with EcoRI generated an intense band of a DNA fragment of about 0.36 kb long. The DNA fragment cloned into pUC19 was used to hybridize with the total rice genomic DNA partially digested with EcoRI. A ladder of bands of DNA fragments with multiplied length of 0.36 kb was observed, demonstrating that this sequence occurs in tandem in the genome. The copy number of the sequence estimated by dot blot hybridization analysis was 2000-3000 copies per haploid genome from callus or seedling of C5924. This sequence was present in other O. sativa cultivars, such as Sasanishiki in 700-900 copies, Koshihikari in 3400-4300, and Nipponbare in 4600-6000 copies. Another rice species, O. glaberrima, also had this sequence in 540-680 copies, but four lines of foxtail millet had none. The DNA fragments containing the repeated sequences in Nipponbare were then cloned into lambda EMBL3, and sequences of nine units consecutively repeated and an AT-rich sequence connected with them in a phage clone could be determined. Each repeating unit showed sequence divergency mostly by substitution of bases in a range from 3% to 7%, when compared with a 355-bp consensus sequence. Analyses of the substituted bases indicate that these are due to spontaneous mutations which occurred at random, after reiteration of a unit sequence by unequal crossing over events. Gene conversion within the repeated sequences might have further diversified their sequences.

Base Sequence↗

PubMed2004

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situ hybridization.

Previously, polymerase chain reaction (PCR) technology has been hampered by its inability to generate quantitative results, a drawback inherent to the high degree of amplification taking place in the reaction. Recently, PCR techniques have been described with the potential of quantifying the amount of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2, and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real-time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Tumor biopsies were collected from 112 patients diagnosed with early breast cancer from January 1990 to March 1994. The samples were analyzed for HER2 DNA amplification by real-time PCR on LightCycler and by FISH and for HER2 protein expression by IHC. Inter-assay variation for HER2 measured by LightCycler was 10% (x =3.1; n=17). Amplification > or = 2 was observed in 19% of the patients. Concordance rates between real-time PCR and the other methods were 91% (IHC) and 92% (FISH). The correlation between real-time PCR and FISH was highly significant (p < 0.001). The "LightCycler-HER2/neu DNA quantification kit" produces results with a high level of reproducibility and its ease of use allows rapid screening for amplification of HER2. In this paper useful information is given on how real-time PCR compares with FISH and IHC. The data show that results obtained for amplification of HER2 by real-time PCR on the LightCycler instrument are comparable to results obtained by IHC and FISH.

Awards and Prizes↗

PubMed1991

Sequence microheterogeneity of the three small subunit ribosomal RNA genes of Babesia bigemina: expression in erythrocyte culture.

Three distinct ribosomal RNA (rRNA) transcription units (rDNA units), designated A, B and C, were identified in the intraerythrocytic protozoan parasite, Babesia bigemina. These rDNA units were cloned, and restriction maps were constructed showing the approximate location of the small and large rRNA coding regions. The arrangement of the genes in the genome and copy number analysis suggests the presence of a single copy of each rDNA unit per haploid genome. The complete nucleotide sequence of the small subunit rRNA coding region (1693 bp) and parts of the 5' and 3' flanking regions were determined for all three units. Units A and B have identical sequences, but unit C differs from units A and B at ten nucleotide positions, two in the small subunit rRNA coding region and four each in the adjacent 5' and 3' flanking regions. The differences in the coding region are confirmed in genomic DNA and RNA from two different isolates of B.bigemina. The RNA of both sequence types is transcribed in parasites from erythrocyte culture, however, the products of gene units A + B accumulate at a ratio of approximately 4:1 compared with the product of unit C.

Animals↗

PubMed2002

Wanda: a database of duplicated fish genes.

Comparative genomics has shown that ray-finned fish (Actinopterygii) contain more copies of many genes than other vertebrates. A large number of these additional genes appear to have been produced during a genome duplication event that occurred early during the evolution of Actinopterygii (i.e. before the teleost radiation). In addition to this ancient genome duplication event, many lineages within Actinopterygii have experienced more recent genome duplications. Here we introduce a curated database named Wanda that lists groups of orthologous genes with one copy from man, mouse and chicken, one or two from tetraploid Xenopus and two or more ancient copies (i.e. paralogs) from ray-finned fish. The database also contains the sequence alignments and phylogenetic trees that were necessary for determining the correct orthologous and paralogous relationships among genes. Where available, map positions and functional data are also reported. The Wanda database should be of particular use to evolutionary and developmental biologists who are interested in the evolutionary and functional divergence of genes after duplication. Wanda is available at http://www.evolutionsbiologie.uni-konstanz.de/Wanda/.

Animals↗

PubMed2003

Molecular assay to generate expression profile of eight estrogen receptor alpha isoform mRNA copy numbers in picogram amounts of total RNA from breast cancer tissues.

The presence of estrogen receptor alpha (ERalpha) is the basis for treating breast cancer patients with targeted molecular therapies that block estrogen stimulation of breast cancer cell division. Currently, the ERalpha presence is determined by microscopically scoring the slides subjected to immunohistochemistry. This method is not quantitative, cannot distinguish between all the known ERalpha isoforms, and requires large amounts of tumor tissue. We describe here a real-time PCR-based molecular approach that can be applied to determine mRNA copies of eight ERalpha isoforms in picogram amounts of total RNA from clinical samples. Each isoform mRNA is quantified using a specific primer pair and a 5'FAM- and 3'TAMARA-labeled probe in comparison with a standard curve constructed with known copy numbers of its respective reverse-transcribed cRNA. Seven alternatively spliced isoforms were quantified using splice-targeted primers. The cRNAs for eight isoforms were generated by in vitro transcription of their respective coding sequences. The sensitivity of detection with reverse-transcribed cRNAs is as low as 100 copies. The devised assays can detect ERalpha cDNAs reverse transcribed from as low as 50-100 pg of total RNA from breast cancer tissues. The applicability of the devised assays for profiling eight ERalpha isoform mRNAs is demonstrated using 6 breast cancer cell lines and 10 breast cancer tissues. It is expected that these assays could be applied to profile ERalpha isoforms in any estrogen-responsive tissues. In addition, these methods could highly facilitate the design of tissue-specific selective estrogen receptor modulators to treat breast cancers and other estrogen-related abnormalities.

Alternative Splicing↗

PubMed2026

Somatic mutations reveal hyperactive Notch signaling in prurigo nodularis.

Prurigo nodularis (PN) is a chronic inflammatory skin disease characterized by pruritic skin nodules of unknown etiology. Little is known about genetic changes in PN pathogenesis, particularly somatic events, which are often implicated in inflammatory conditions. We thus performed whole-exome sequencing on 54 lesional and nonlesional skin biopsies from 17 patients with PN and 10 patients with atopic dermatitis (AD) for comparison. Somatic mutational analysis revealed that PN lesional skin harbors recurrent somatic mutations in fibrotic, neurotropic, and cancer-associated genes that are absent in adjacent PN nonlesional skin. Nonsynonymous mutations were most frequently present in NOTCH1 and the Notch signaling pathway, a key regulator of cellular proliferation and tissue fibrosis. In contrast, NOTCH1 mutations were absent in AD. Somatic copy-number analysis, combined with expression data, identified recurrently deleted and downregulated genes in PN lesional skin, which are associated with axonal guidance and extension. Follow-up immunofluorescence validation demonstrated increased NOTCH1 expression in PN lesional skin fibroblasts and increased Notch signaling in PN lesional dermis. Finally, a multicenter analysis revealed increased risk of NOTCH1-associated diseases in patients with PN. In characterizing the somatic landscape of PN, this study highlights the potential role of Notch pathway dysregulation in PN pathogenesis and fibrosis.

Humans↗

PubMed2006

Sub-speciating Campylobacter jejuni by proteomic analysis of its protein biomarkers and their post-translational modifications.

We have identified several protein biomarkers of three Campylobacter jejuni strains (RM1221, RM1859, and RM3782) by proteomic techniques. The protein biomarkers identified are prominently observed in the time-of-flight mass spectra (TOF MS) of bacterial cell lysate supernatants ionized by matrix-assisted laser desorption/ionization (MALDI). The protein biomarkers identified were: DNA-binding protein HU, translation initiation factor IF-1, cytochrome c553, a transthyretin-like periplasmic protein, chaperonin GroES, thioredoxin Trx, and ribosomal proteins: L7/L12 (50S), L24 (50S), S16 (30S), L29 (50S), and S15 (30S), and conserved proteins similar to strain NCTC 11168 proteins Cj1164 and Cj1225. The protein biomarkers identified appear to represent high copy, intact proteins. The significant findings are as follows: (1) Biomarker mass shifts between these strains were due to amino acid substitutions of the primary polypeptide sequence and not due to changes in post-translational modifications (PTMs). (2) If present, a PTM of a protein biomarker appeared consistently for all three strains, which supported that the biomarker mass shifts observed between strains were not due to PTM variability. (3) The PTMs observed included N-terminal methionine (N-Met) cleavage as well as a number of other PTMs. (4) It was discovered that protein biomarkers of C. jejuni (as well as other thermophilic Campylobacters) appear to violate the N-Met cleavage rule of bacterial proteins, which predicts N-Met cleavage if the penultimate residue is threonine. Two protein biomarkers (HU and 30S ribosomal protein S16) that have a penultimate threonine residue do not show N-Met cleavage. In all other cases, the rule correctly predicted N-Met cleavage among the biomarkers analyzed. This exception to the N-Met cleavage rule has implications for the development of bioinformatics algorithms for protein/pathogen identification. (5) There were fewer biomarker mass shifts between strains RM1221 and RM1859 compared to strain RM3782. As the mass shifts were due to the frequency of amino acid substitutions (and thus underlying genetic variations), this suggested that strains RM1221 and RM1859 were phylogenetically closer to one another than to strain RM3782 (in addition, a protein biomarker prominent in the spectra of RM1221 and RM1859 was absent from the RM3782 spectrum due to a nonsense mutation in the gene of the biomarker). These observations were confirmed by a nitrate reduction test, which showed that RM1221 and RM1859 were C. jejuni subsp. jejuni whereas RM3782 was C. jejuni subsp. doylei. This result suggests that detection/identification of protein biomarkers by pattern recognition and/or bioinformatics algorithms may easily subspeciate bacterial microorganisms. (6) Finally, the number and variation of PTMs detected in this relatively small number of protein biomarkers suggest that bioinformatics algorithms for pathogen identification may need to incorporate many more possible PTMs than suggested previously in the literature.

Amino Acid Sequence↗