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PubMed2005

Chemostat culture characterization of Escherichia coli mutant strains metabolically engineered for aerobic succinate production: a study of the modified metabolic network based on metabolite profile, enzyme activity, and gene expression profile.

Various Escherichia coli mutant strains designed for succinate production under aerobic conditions were characterized in chemostat. The metabolite profiles, enzyme activities, and gene expression profiles were studied to better understand the metabolic network operating in these mutant strains. The most efficient succinate producing mutant strain HL27659k was able to achieve a succinate yield of 0.91 mol/mol glucose at a dilution rate of 0.1/h. This strain has the five following mutations: sdhAB, (ackA-pta), poxB, iclR, and ptsG. Four other strains involved in this study were HL2765k, HL276k, HL2761k, and HL51276k. Strain HL2765k has mutations in sdhAB, (ackA-pta), poxB and iclR, strain HL276k has mutations in sdhAB, (ackA-pta) and poxB, strain HL2761k has mutations in sdhAB, (ackA-pta), poxB and icd, and strain HL51276k has mutations in iclR, icd, sdhAB, (ackA-pta) and poxB. Enzyme activity data showed strain HL27659k has substantially higher citrate synthase and malate dehydrogenase activities than the other four strains. The data also showed that only iclR mutation strains exhibited isocitrate lyase and malate synthase activities. Gene expression profiles also complemented the studies of enzyme activity and metabolites from chemostat cultures. The results showed that the succinate synthesis pathways engineered in strain HL27659k were highly efficient, yielding succinate as the only major product produced under aerobic conditions. Strain HL27659k was the only strain without pyruvate accumulation, and its acetate production was the least among all the mutant strains examined.

Aerobiosis↗

PubMedDate not supplied

SELDI-TOF serum profiling for prognostic and diagnostic classification of breast cancers.

Surface enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry has emerged as a successful tool for serum based detection and differentiation of many cancer types, including breast cancers. In this study, we have applied the SELDI technology to evaluate three potential applications that could extend the effectiveness of established procedures and biomarkers used for prognostication of breast cancers. Paired serum samples obtained from women with breast cancers prior to surgery and post-surgery (6-9 mos.) were examined. In 14/16 post-treatment patients, serum protein profiles could be used to distinguish these samples from the pre-treatment cancer samples. When compared to serum samples from normal healthy women, 11 of these post-treatment samples retained global protein profiles not found in healthy women, including five low-mass proteins that remained elevated in both pre-treatment and post-treatment serum groups. In another pilot study, serum profiles were compared for a group of 30 women who were known BRCA-1 mutation carriers, half of whom subsequently developed breast cancer within three years of the sample procurement. SELDI protein profiling accurately classified 13/15 women with BRCA-1 breast cancers from the 15 non-cancer BRCA-1 carriers. Additionally, the ability of SELDI to distinguish between the serum profiles from sentinel lymph node positive and sentinel lymph node negative patients was evaluated. In sentinel lymph node positive samples, 22/27 samples were correctly classified, in comparison to the correct classification of 55/71 sentinel lymph node negative samples. These initial results indicate the utility of protein profiling approaches for developing new diagnostic and prognostic assays for breast cancers.

Adult↗

PubMed2000

Laminar specific loss of isocortical presenilin 1 immunoreactivity in Alzheimer's disease. Correlations with the amyloid load and the density of tau-positive neurofibrillary tangles.

Presenilin 1 has been shown to be mutated in a high proportion of cases of familial Alzheimer's disease. Immunoreactive epitopes of the protein have been found mainly in neurones devoid of neurofibrillary tangles - an observation that has led to the conclusion that presenilin 1 could have a protective role. In this study, the relationship between deposits of Abeta peptide (both the 40 and 42 isoforms), tau positive neurofibrillary tangles and presenilin 1-positive neuronal profiles were analysed in three cases of presenilin 1 mutation, four cases of sporadic Alzheimer's disease and five controls. Immunohistochemistry was performed in a sample from the supramarginal gyrus. The proportion of volume occupied by the Abeta1-40 and Abeta1-42 deposits (amyloid load) was evaluated by a point-counting technique. Tau-positive neurofibrillary tangles, and presenilin 1-positive neuronal profiles were directly counted. The location of the lesions in the thickness of the cortex was recorded. The density of PS1-positive neuronal profiles in Alzheimer's disease cases was lower than in the controls. The deficit was significant only in the upper layers of the cortex. The density of presenilin 1 neuronal profiles was negatively correlated with Abeta1-40 and Abeta1-42 loads, and with the density of tau-positive neurofibrillary tangles. Multivariate analysis showed that the Abeta1-42 load was the best determinant of the decrease in presenilin 1-positive neuronal profiles. Presenilin 1-positive neurones appear to be lost rather than protected in the course of Alzheimer disease.

Adult↗

PubMed1993

The ovine Booroola fecundity gene (FecB) is linked to markers from a region of human chromosome 4q.

The autosomal Booroola fecundity gene (FecB) mutation in sheep increases ovulation rate and litter size, with associated effects on ovarian physiology and hormone profiles. Analysis of segregation in twelve families (379 female progeny) identified linkage between the mutation, two microsatellite markers (OarAE101 and OarHH55, Zmax > 9.0) and epidermal growth factor (EGF) from human chromosome 4q25 (Zmax > 3.0). The marker OarAE101 was linked to secreted phosphoprotein 1 (SPP1, which maps to chromosome 4q21-23 in man) in the test pedigrees and independent families (Zmax > 9.7). The identification of linkage between the FecB mutation and markers from human chromosome 4q is an important step towards further understanding the control of ovulation rates in mammals.

Animals↗

PubMed2006

Differential activation of IFN regulatory factor (IRF)-3 and IRF-5 transcription factors during viral infection.

Members of the IFN regulatory factor (IRF) family regulate gene expression critical to immune response, hemopoiesis, and proliferation. Although related by homology at their N-terminal DNA-binding domain, they display individual functional properties. The distinct properties result from differences in regulated expression, response to activating signals, and interaction with DNA regulatory elements. IRF-3 is expressed ubiquitously and is activated by serine phosphorylation in response to viral infection or TLR signaling. Evidence indicates that the kinases TANK-binding kinase 1 and inhibitor of NF-kappaB kinase-epsilon specifically phosphorylate and thereby activate IRF-3. We evaluated the contribution of another member of the IRF family, IRF-5, during viral infection since prior studies provided varied results. Analysis of phosphorylation, nuclear translocation, dimerization, binding to CREB-binding protein, recognition of DNA, and induction of gene expression were used comparatively with IRF-3 as a measure of IRF-5 activation. IRF-5 was not activated by viral infection; however, expression of TANK-binding kinase 1 or inhibitor of NF-kappaB kinase-epsilon did provide clear activation of IRF-5. IRF-5 is therefore distinct in its activation profile from IRF-3. However, similar to the biological effects of IRF-3 activation, a constitutively active mutation of IRF-5 promoted apoptosis. The apoptosis was inhibited by expression of Bcl-x(L) but not a dominant-negative mutation of the Fas-associated death domain. These studies support the distinct activation profiles of IRF-3 in comparison to IRF-5, but reveal a potential shared biological effect.

Amino Acid Sequence↗

PubMed1997

Novel point mutation in the uroporphyrinogen III synthase gene causes congenital erythropoietic porphyria of a Japanese family.

The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in a member of a Japanese family. This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Günther's disease. The patient was homozygous for a novel missense mutation: a G to T transition of nucleotide 7 that predicted a valine to phenylalanine substitution at residue 3 (V3F). The parents were heterozygous for the same mutation. The loss of UROIIIS activity was verified by an in vitro assay system. The corresponding mutated protein was expressed in Escherichia coli and no residual activity was observed. Further studies are needed to determine whether the mutations of the UROIIIS gene (UROS) have a specific profile in Japan compared to European or American countries.

Adult↗

PubMed2006

A third approach to gene prediction suggests thousands of additional human transcribed regions.

The identification and characterization of the complete ensemble of genes is a main goal of deciphering the digital information stored in the human genome. Many algorithms for computational gene prediction have been described, ultimately derived from two basic concepts: (1) modeling gene structure and (2) recognizing sequence similarity. Successful hybrid methods combining these two concepts have also been developed. We present a third orthogonal approach to gene prediction, based on detecting the genomic signatures of transcription, accumulated over evolutionary time. We discuss four algorithms based on this third concept: Greens and CHOWDER, which quantify mutational strand biases caused by transcription-coupled DNA repair, and ROAST and PASTA, which are based on strand-specific selection against polyadenylation signals. We combined these algorithms into an integrated method called FEAST, which we used to predict the location and orientation of thousands of putative transcription units not overlapping known genes. Many of the newly predicted transcriptional units do not appear to code for proteins. The new algorithms are particularly apt at detecting genes with long introns and lacking sequence conservation. They therefore complement existing gene prediction methods and will help identify functional transcripts within many apparent "genomic deserts."

Algorithms↗

PubMed2006

The N-terminal domain of the human eIF2beta subunit and the CK2 phosphorylation sites are required for its function.

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2alpha evidenced the direct involvement of this protein kinase in eIF2beta phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2beta or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2beta phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2beta also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2beta S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Delta2-138 N-terminal-truncated form of eIF2beta (eIF2beta-CT). Mutation at Ser2 and Ser67 did not affect eIF2beta integrating into the eIF2 trimer or being able to complex with eIF5 and CK2alpha. The eIF2beta-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2beta slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2beta S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2beta-CT form, being detectable at doses where eIF2beta and eIF2beta S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2beta for its function in mammals.

Amino Acid Sequence↗

PubMed2006

OPTN gene: profile of patients with glaucoma from India.

PURPOSE: Optineurin gene (OPTN) mutations are reported in primary open angle glaucoma patients (POAG) from different populations. The coding and noncoding regions of OPTN were screened for mutations in 100 Indian high tension glaucoma patients (HTG). The frequency of the OPTN M98K mutation in an additional 120 patients (70 HTG and 50 normal tension glaucoma [NTG]) was analyzed by restriction enzyme digestion. METHODS: The HTG patients (about 40 years of age) were characterized by open angles on gonioscopy, with raised intraocular pressure (IOP) more than 21 mmHg (<21 mmHg on office diurnal phasing for NTG), and typical glaucomatous disc changes with corresponding visual field defects in the absence of any secondary cause. One hundred HTG patients and controls were screened for OPTN mutations by direct sequencing using an ABI prism 310/3100 Avant genetic analyzer. The M98K status was analyzed by restriction enzyme digestion with StuI. A genotype/phenotype correlation was also attempted for OPTN sequence alterations with clinical parameters such as age at diagnosis, intraocular pressure, cup:disc ratio, etc. The putative change in the transcription factor binding site for the IVS7 +24G>A polymorphism was attempted with AliBaba software (version 2.1). RESULTS: Six sequence alterations were observed in the 100 POAG patients by direct sequencing. The M98K substitution was observed in a total of 10 patients (7/170 HTG and 3/50 NTG) contributing to 4.1% in HTG and 6% in the NTG group and not in the controls. The IVS7+24G>A nucleotide change showed a significant difference in the HTG group (7/100) when compared to the control group (0/100) and found to be associated with increased IOP at diagnosis (p=0.03). The IVS7+24G>A polymorphism resulted in the creation of binding sites for transcription factors NF-1 and CPE that were not present in the wild type. CONCLUSIONS: The current study suggests a possible role of SNPs rather than mutations in OPTN in POAG pathology in the Indian population.

Adenine↗

PubMed2003

A panoramic view of yeast noncoding RNA processing.

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.

Cells, Cultured↗

PubMed2004

Segregation analysis in cystic fibrosis at-risk family demonstrates that the M348K CFTR mutation is a rare innocuous polymorphism.

OBJECTIVE: Cystic fibrosis (CF; OMIM# 219700) is caused by mutation in the CF transmembrane regulator (CFTR) gene. We investigate whether the (paternal) M348K mutation is a benign polymorphism or a disease-causing mutation in a patient clinically affected with CF, with the second (maternal) CFTR allele identified as N1303K. METHODS: The patient and his father were studied for the presence of mutations in the CFTR gene using the DHPLC system to analyze all CFTR exons. Amplicons showing an abnormal elution profile were sequenced. RESULTS: The CFTR gene from the healthy father has two mutations, M348K and G1244E. The affected son inherited only the G1244E paternal mutation from his father, and hence the two paternal mutations are trans and do not occur in the same CFTR gene. The patient's genotype is G1244E(paternal)/N1303K(maternal). This information was used to study an ongoing pregnancy of the couple, where the fetus inherited the same genotype as the affected proband and therefore is affected. CONCLUSION: M348K in the CFTR gene is not a mutation causing CF, but a rare polymorphism. These data are important for genetic counseling and prenatal diagnosis and illustrate the importance of full sequence data when studying rare mutations.

Alleles↗

PubMed2000

Polymorphisms in dopamine receptors: what do they tell us?

Many genetic studies have focussed on dopamine receptors and their relationship to neuropsychiatric disease. Schizophrenia, bipolar disorder, and substance abuse have been the most studied, but no conclusive linkage or association has been found. The possible influence of dopamine receptor variants on drug response has not received as much attention. While there is some evidence that polymorphisms and mutations in dopamine receptors can alter functional activity and pharmacological profiles, no conclusive data link these gene variants to drug response or disease. The lack of unequivocal findings may be related, in part, to the subtle changes in receptor pharmacology that these polymorphisms and mutations mediate. These subtle effects may be obscured by the influence of genes controlling drug metabolism and kinetics. Further insight into the pharmacogenetics of dopamine receptors may require not just more studies, but novel approaches to the study of complex genetic traits and diseases.

Animals↗

PubMedDate not supplied

Plasmodium falciparum: in vitro activity of sulfadoxine and dapsone in field isolates from Kenya: point mutations in dihydropteroate synthase may not be the only determinants in sulfa resistance.

We have determined the relationship between point mutations in the gene that encodes the sulfa target, dihydropteroate synthase (DHPS) and the chemosensitivity profile to sulfadoxine and dapsone in 67 isolates from Kilifi, Kenya. We assessed the presence of mutations at codons 436, 437, 540, 581, and 613 of dhps. The results showed that the dhps genotype had a strong influence on the sensitivity to sulfadoxine and dapsone, but that the correlation was far from perfect. Eleven isolates carried a wild-type dhps allele, but were resistant to sulfadoxine (IC(50) values >10 microg/ml), and 4/28 isolates were classed as sensitive to sulfadoxine (IC(50) values <10 microg/ml), but carried a triple mutant (436/437/613) allele of dhps. These data show that in low folate medium in vitro, the dhps genotype alone did not account completely for sulfadoxine or dapsone resistance; other factors such as the utilisation of exogenous folate must also be considered.

Alleles↗

PubMed2000

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas.

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (V(H)) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Biopsy↗

PubMed2004

Role of serine 10 phosphorylation in p27 stabilization revealed by analysis of p27 knock-in mice harboring a serine 10 mutation.

The inhibition of cyclin-dependent kinase activity by p27 contributes to regulation of cell cycle progression. Serine 10 is the major phosphorylation site of p27, and its phosphorylation has been shown to affect the stability and nuclear export of p27 at the G0-G1 transition in transfected cultured cells. To investigate the physiological relevance of p27 phosphorylation on Ser10, we generated p27 "knock-in" mice that harbor an S10A mutation in this protein. Mice homozygous for the mutation (p27(S10A/S10A) mice) were normal in body size, but the abundance of p27 was decreased in many organs, including brain, thymus, spleen, and testis. The stability of p27 in G0 phase was markedly reduced in lymphocytes of p27(S10A/S10A) mice compared with that in wild-type cells, whereas p27 stability in S phase was similar in cells of the two genotypes. The degradation of p27 in cells of the mutant mice at G0 phase was prevented by a proteasome inhibitor. These data indicate that the physiological role of p27 phosphorylation on Ser10 is to stabilize the protein in G0 phase. Unexpectedly, the nuclear export of p27 at the G0-G1 transition occurred normally in p27(S10A/S10A) mouse embryonic fibroblasts, indicating that phosphorylation of Ser10 is dispensable for this process.

Active Transport, Cell Nucleus↗

PubMed2005

Coexpression of erythropoietin and its receptor in endolymphatic sac tumors.

OBJECT: Von Hippel-Lindau (VHL) disease is characterized by multiple tumors in specific organs. The cell of origin and the reason for the particular organ distribution of the tumors remains unknown. Endolymphatic sac tumor (ELST) is one of the lesions associated with VHL disease. Data from previous studies of VHL disease-associated hemangioblastomas (HBs) and renal cell carcinomas (RCCs) have indicated that VHL gene deficiency causes coexpression of erythropoietin (Epo) and its receptor (Epo-R), which facilitates tumor growth. METHODS: The authors studied ELSTs from five patients with VHL germline mutations. Analysis of the five ELST samples revealed loss of the wild-type allele, consistent with Knudson's two-hit hypothesis for tumorigenesis. All five ELST specimens were characterized microscopically and by immunohistochemical analysis. Coexpression of Epo and Epo-R was found in all five tumors on immunohistochemical studies and confirmed through reverse transcription-polymerase chain reaction and Western blot analysis. CONCLUSIONS: Expression of Epo appears to be a result of VHL gene deficiency, whereas the simultaneous coexpression of Epo-R may reflect a developmental mechanism of tumorigenesis. Coexpression of Epo and Epo-R in ELSTs together with the morphological and genetic similarities of these lesions with other VHL disease-associated tumors indicates that VHL disease-associated tumors in different organs share common pathogenetic pathways.

Adult↗

PubMed2006

Down-regulation of key virulence factors makes the Salmonella enterica serovar Typhimurium rfaH mutant a promising live-attenuated vaccine candidate.

Mutants of Salmonella enterica serovar Typhimurium that lack the transcriptional regulator RfaH are efficient as live oral vaccines against salmonellosis in mice. We show that the attenuation of the vaccine candidate strain is associated with reduced net growth in epithelial and macrophage cells. In order to identify the relevant RfaH-dependent genes, the RfaH regulon was determined with S. enterica serovars Enteritidis and Typhimurium using whole-genome Salmonella microarrays. As well as impacting the expression of genes involved in lipopolysaccharide (LPS) core and O-antigen synthesis, the loss of RfaH results in a marked down-regulation of SPI-4 genes, the flagellum/chemotaxis system, and type III secretion system 1. However, a proportion of these effects could have been the indirect consequence of the altered expression of genes required for LPS biosynthesis. Direct and indirect effects of the rfaH mutation were dissociated by genome-wide transcriptional profiling of a structural deep-rough LPS mutant (waaG). We show that truncation of LPS itself is responsible for the decreased intracellular yield observed for DeltarfaH strains. LPS mutants do not differ in replication ability; rather, they show increased susceptibility to antimicrobial peptides in the intracellular milieu. On the other hand, evidence that deletion of rfaH, as well as some other genes involved in LPS biosynthesis, results in enhanced invasion of various mammalian cells is shown. Exposure of common minor antigens in the absence of serovar-specific antigens might be responsible for the observed cross-reactive nature of the elicited immune response upon vaccination. Increased invasiveness of the Salmonella rfaH mutant into antigen-presenting cells, combined with increased intracellular killing and the potential for raising a cross-protective immune response, renders the rfaH mutant an ideal vaccine candidate.

Animals↗

PubMed2005

Gene expression profiling reveals an inflammatory process in the anx/anx mutant mice.

Anorexia (anx) is a recessive mutation that causes lethal starvation in homozygous mice. Studies of anx/anx mice hypothalamus have shown abnormalities in the orexigenic (NPY/AGRP neurons) and the anorexigenic (POMC/CART neurons) pathways. By gene expression profiling using cDNA and oligonucleotide microarrays, we have shown that a surexpression of genes involved in inflammatory process occurred in anx mice hypothalamus. This inflammatory process could be the cause of the anorexia phenotype observed in these mice.

Animals↗