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PubMed2001

Quality of life influenced by primary surgical treatment for stage I-III breast cancer-long-term follow-up of a matched-pair analysis.

BACKGROUND: Breast-conserving therapy has been demonstrated to be just as safe and a less disruptive experience compared with mastectomy for surgically manageable breast cancer. There is, however, no agreement in the literature about the impact of these procedures on several important aspects of quality of life (QOL). The purpose of the present study is to compare the long-term impact of these two surgical approaches on QOL in patients with identical tumor stages and to suggest possible shortcomings of the standard QOL questionnaires. METHOD: Between August 1999 and May 2000, QOL questionnaires were answered by 152 pair-matched patients at the I. Frauenklinik, Ludwig-Maximilians University Munich, as part of routine follow-up examinations. The pairs of patients, each consisting of one patient after mastectomy and one after breast conservation, were selected according to the highest degree of equivalence in tumor stage. All patients had been initially treated for stage I-III breast cancer without evidence of distant metastases. The QOL was evaluated by using the QLQ-C30 questionnaire version 2.0 of the EORTC Study Group on Quality of Life. We formulated seven additional questions about the patients' satisfaction with the primary surgical treatment modality as viewed from their current perspective. The QOL questionnaires were answered after a median interval of 46 months following primary treatment. RESULTS: Tumor stage, prognostic factors, and adjuvant systemic treatment were well balanced between the two groups. No differences between the two groups were observed in terms of all QOL items measured by the QLQ-C30. Our additional questions, however, revealed that patients in the mastectomy group were less satisfied with the cosmetic result of their primary operation (P < .0001), were more likely to feel basic changes in their appearance (P < .0001), and were more likely to be emotionally stressed by these facts (P < .0001). From their perspective at the time of completing the questionnaires, 11 patients in the mastectomy group (15%) would decide differently about the surgical treatment modality, compared with only 3 patients (4%) in the breast conservation group (P = .025). CONCLUSION: While the primary surgical treatment modality seems to have no long-term impact on general QOL, certain body-image-related problems may be caused by mastectomy. Standard measuring instruments for QOL may fail to detect differences in satisfaction and adaptation with the primary surgical treatment modality.

Breast Neoplasms↗
PubMed1987

Extensive structural differences between genes for the alpha 1 and alpha 2 chains of type IV collagen despite conservation of coding sequences.

Analysis of the structure of the 3'-end of the human alpha 2(IV) gene demonstrated that the alpha 1(IV) and alpha 2(IV) genes have diverged extensively in spite of the apparent homology of the respective gene products. The NC-1 domain and the 3'-untranslated region are encoded by three exons in the alpha 2(IV) gene but five exons in the alpha 1(IV) gene. The two introns present in the NC-1 domain coding part of the alpha 2(IV) gene had the same location as two of the introns of the alpha 1(IV) gene. The junction exon in the alpha 2(IV) gene contains 53 bp coding for Gly-X-Y sequences whereas there are 71 bp in the alpha 1(IV) gene. Three other Gly-X-Y coding exons studied from the human alpha 2(IV) gene have sizes that differ from corresponding exons in the alpha 1(IV) gene and only one intron location matches here between the two genes. None of the exons studied has 54 bp or multiples thereof.

Animals↗
PubMed2006

Detection and phylogenic analysis of one anthrax virulence plasmid pXO1 conservative open reading frame ubiquitous presented within Bacillus cereus group strains.

The presence of one of the anthrax virulence plasmid pXO1 conserved fragments was analyzed in 24 Bacillus cereus and B. thuringiensis strains, including 6 B. thuringiensis subspecies, by polymerase chain reactions. Twelve out of 24 strains showed PCR-positive for an ORF101 homologous sequence. Two pXO1-ORF101-like fragments from a B. cereus B-4ac and a commercial B. thuringiensis kurstaki HD1 were cloned, sequenced and expressed in Escherichia coli. Toxicity assays revealed that the product encoded by the pXO1-ORF101-like fragment had no impact on either Vero cells or Chinese Hamster Ovary cells, suggesting that this fragment probably not contribute to enterotoxic activity. Sequence alignment of the pXO1-ORF101 from three Bacillus anthracis and ORF101-like fragments from other 12 B. cereus group isolates indicated high identity (more than 90%) and the presence of subgroup- and strain-specific SNPs among these fragments.

Bacillus cereus↗
PubMed2005

Radiochemotherapy in the conservative treatment of anal canal carcinoma: retrospective analysis of results and radiation dose effectiveness.

PURPOSE: This retrospective analysis reports the results on patients with anal canal carcinoma treated by combined radiotherapy and chemotherapy. METHODS AND MATERIALS: Between March 1993 and December 2001, 43 patients with anal canal carcinoma were treated with radiochemotherapy at the Hospital do Cancer A.C. Camargo. Stage distribution was as follows: I, 3 (7%); II, 23 (53.5%); IIIA, 8 (18.6%); and IIIB, 9 (21%). The median age was 56 years (range, 36-77 years) with most patients being women (4:1). External radiotherapy (RT) was delivered at the whole pelvis followed by a boost at the primary tumor. The median dose of RT at the whole pelvis and at the primary tumor was 45 Gy and 55 Gy, respectively. Chemotherapy was carried out during the first and last 4 days of RT with continuous infusion of 5-fluorouracil (1000 mg/m(2)) and bolus mitomycin C (10 mg/m(2)). Median overall treatment time was 51 days (range, 30-129 days). Thirty-four patients (79%) did not receive elective RT at the inguinal region. Patient's age, tumor stage, overall treatment time, and RT dose at primary tumor were variables analyzed for survival and local control. RESULTS: Median follow-up time was 42 months (range, 4-116 months). Overall survival and colostomy-free survival at 5 years was 68% and 52%, respectively. Overall survival according to clinical stage was as follows: I, 100%; II, 82%; IIIA, 73%; and IIIB, 18% (p = 0.0049). Complete response was observed in 40 patients (93%). Local recurrence occurred in 9 (21%) patients, and of these, 6 were rescued by surgery. Local control with a preserved sphincter was observed in 34 patients (79%). According to the RT dose, local control was higher among patients who received more than 50 Gy at primary tumor (86.5% vs. 34%, p = 0.012). Inguinal failure was observed in 5 patients (15%) who did not receive inguinal elective RT. Distant metastasis was observed in 11 patients (25.6%). Temporary interruption of the treatment as a result of acute toxicity was necessary in 12 patients (28%). Four patients developed mild chronic complications. CONCLUSIONS: This analysis suggests that the treatment scheme employed was effective for anal sphincter preservation and local control; however, the incidence of distant metastases was relatively high. The clinical stage was the main prognostic factor for overall survival. Local control was higher in patients treated with doses of more than 50 Gy at primary tumor. The high incidence of inguinal failure implies the need for elective RT in this region.

Adult↗
PubMed1996

Randomised controlled trial of conservation therapy for breast cancer: 6-year analysis of the Scottish trial. Scottish Cancer Trials Breast Group.

BACKGROUND: To determine whether, when primary breast cancer is treated by local excision supported by systemic therapy appropriate to the oestrogen receptor status (ER) of the tumour, local radiotherapy can be avoided. METHODS: We carried out a randomised controlled trial in 585 patients aged less than 70 years with primary breast cancers of 4 cm or less in size in four specialist units and seven other hospitals in Scotland. After local excision of the tumour (1 cm margin) and an axillary lymph-node clearance or sample, all patients received systemic therapy with oral tamoxifen 20 mg daily or six 3-weekly intravenous bolus injections of cyclophosphamide 600 mg, methotrexate 50 mg, and fluorouracil 600 mg per m2, depending upon the ER concentration in the primary tumour. Patients were then randomly allocated to postoperative radical radiotherapy (50 Gy to breast with boost to the tumour bed) or to no further local treatment. The median follow-up of living patients was 5.7 years. The primary analysis was by intention to treat but since some patients did not receive systemic therapy appropriate to their ER status, a subsidiary analysis was restricted to 464 patients in whom all details of the protocol had been observed. FINDINGS: In the primary analysis survival was equal in the radiotherapy and non-radiotherapy groups (hazard ratio [HR] 0.98, 95% CI 0.67-1.44). Event-free survival showed an advantage in the irradiated patients (HR 0.54, 95% CI 0.39-0.74), largely due to fewer loco-regional relapses (HR 0.20, 95% CI 0.12-0.33). The relapse rate in the ipsilateral breast was 24.5% in the non-irradiated group and 5.8% following breast irradiation. The subsidiary analysis confirmed these findings and indicated the advantage of radiotherapy irrespective of ER concentration. There was a non-significant trend towards fewer distant metastases in the irradiated group. INTERPRETATION: After local excision of a primary breast cancer, we conclude that radiotherapy to the residual breast tissue is advisable even when selective adjuvant systemic therapy is given.

Adult↗
PubMed2006

Stereochemical analysis of the functional significance of the conserved inverted CCAAT and TATA elements in the rat bone sialoprotein gene promoter.

Basal transcription of the bone sialoprotein gene is mediated by highly conserved inverted CCAAT (ICE; ATTGG) and TATA elements (TTTATA) separated by precisely 21 nucleotides. Here we studied the importance of the relative position and orientation of the CCAAT and TATA elements in the proximal promoter by measuring the transcriptional activity of a series of mutated reporter constructs in transient transfection assays. Whereas inverting the TTTATA (wild type) to a TATAAA (consensus TATA) sequence increased transcription slightly, transcription was reduced when the flanking dinucleotides were also inverted. In contrast, reversing the ATTGG (wild type; ICE) to a CCAAT (RICE) sequence caused a marked reduction in transcription, whereas both transcription and NF-Y binding were progressively increased with the simultaneous inversion of flanking nucleotides (f-RICE-f). Reducing the distance between the ICE and TATA elements produced cyclical changes in transcriptional activity that correlated with progressive alterations in the relative positions of the CCAAT and TATA elements on the face of the DNA helix. Minimal transcription was observed after 5 nucleotides were deleted (equivalent to approximately one half turn of the helix), whereas transcription was fully restored after deleting 10 nucleotides (approximately one full turn of the DNA helix), transcriptional activity being progressively lost with deletions beyond 10 nucleotides. In comparison, when deletions were made with the ICE in the reversed (f-RICE-f) orientation transcriptional activity was progressively lost with no recovery. These results show that, although transcription can still occur when the CCAAT box is reversed and/or displaced relative to the TATA box, the activity is dependent upon the flexibility of the intervening DNA helix needed to align the NF-Y complex on the CCAAT box with preinitiation complex proteins that bind to the TATA box. Thus, the precise location and orientation of the CCAAT element is necessary for optimizing basal transcription of the bone sialoprotein gene.

Animals↗
PubMed1998

Structural and functional analysis of mouse Msx1 gene promoter: sequence conservation with human MSX1 promoter points at potential regulatory elements.

Vertebrate Msx genes are related to one of the most divergent homeobox genes of Drosophila, the muscle segment homeobox (msh) gene, and are expressed in a well-defined pattern at sites of tissue interactions. This pattern of expression is conserved in vertebrates as diverse as quail, zebrafish, and mouse in a range of sites including neural crest, appendages, and craniofacial structures. In the present work, we performed structural and functional analyses in order to identify potential cis-acting elements that may be regulating Msx1 gene expression. To this end, a 4.9-kb segment of the 5'-flanking region was sequenced and analyzed for transcription-factor binding sites. Four regions showing a high concentration of these sites were identified. Transfection assays with fragments of regulatory sequences driving the expression of the bacterial lacZ reporter gene showed that a region of 4 kb upstream of the transcription start site contains positive and negative elements responsible for controlling gene expression. Interestingly, a fragment of 130 bp seems to contain the minimal elements necessary for gene expression, as its removal completely abolishes gene expression in cultured cells. These results are reinforced by comparison of this region with the human Msx1 gene promoter, which shows extensive conservation, including many consensus binding sites, suggesting a regulatory role for them.

Animals↗
PubMed2004

Rounding up active cis-elements in the triple C corral: combining conservation, cleavage and conformation capture for the analysis of regulatory gene domains.

Identification and functional analysis of potential cis-regulatory elements is a laborious process that often depends on removing putative elements from their natural context to study their activity. While such methods provide valuable information about the isolated element, they disregard the potential role of an element's interaction(s) with other regulatory sequences and the three-dimensional structure of an active gene locus. Here, two novel methods are discussed--chromosome conformation capture (3C) and RNA-TRAP--that can be used to detect interactions between distal regulatory sites and which thus indicate the chromosomal conformation that is adopted by a gene locus in various states of transcriptional activity. Combined with comparative genomics and traditional DNase I hypersensitive site mapping, these methods form a powerful approach for the study of the mechanisms of long-range transcriptional regulation.

Animals↗
PubMed1999

The RNA-binding properties of SMN: deletion analysis of the zebrafish orthologue defines domains conserved in evolution.

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder that results in the degeneration of spinal motor neurons. SMA is caused by alterations of the survival motor neuron ( SMN ) gene which encodes a novel protein of hitherto unclear function. The SMN protein associates with ribonucleoprotein particles involved in RNA processing and exhibits an RNA-binding capacity. We have isolated the zebrafish Danio rerio and nematode Caenorhabditis elegans orthologues and have found that the RNA-binding capacity is conserved across species. Purified recombinant SMN proteins from both species showed selectivity to poly(G) homopolymer RNA in vitro, similar to that of the human protein. Studying deletions of the zebrafish SMN protein, we defined an RNA-binding element in exon 2a, which is highly conserved across species, and revealed that its binding activity is modulated by protein domains encoded by exon 2b and exon 3. Finally, the deleted recombinant zebrafish protein mimicking an SMA frameshift mutation showed a dramatic change in vitro in the formation of the RNA-protein complexes. These observations indicate that the RNA-binding capacity of SMN is an evolutionarily conserved function and further support the view that defects in RNA metabolism most likely account for the pathogenesis of SMA.

Amino Acid Sequence↗
PubMed1999

Functional analysis of IgA antibodies specific for a conserved epitope within the M protein of group A streptococci from Australian Aboriginal endemic communities.

The mucosa is one of the initial sites of group A streptococcal (GAS) infection and salivary IgA (sIgA) is thought to be critical to immunity. However, the target epitopes of sIgA and the function of sIgA in GAS immunity, in particular the role of accessory cells and complement, is largely unknown. We studied the aquisition and the function of sIgA specific for a conserved region epitope, p145 (sequence: LRRDLDASREAKKQVEKALE) of the M protein. Peptide 145-specific sIgA is highly prevalent within an Aboriginal population living in an area endemic for GAS and acquisition of p145-specific sIgA increases with age, consistent with a role for such antibodies in immunity to GAS. Human sIgA and IgG specific for p145 were affinity purified and shown to opsonize M5 GAS in vitro. Opsonization could be specifically inhibited by the addition of free p145 to the antibodies during assay. Opsonization of GAS was totally dependent on the presence of both complement and polymorphonuclear leukocytes, and, moreover, affinity-purified p145-specific sIgA was shown to fix complement in the presence of M5 GAS. These data show that mucosal IgA to this conserved region peptide within the M protein has an important role in human immunity against GAS and may be useful in a broad-based cross-protective anti-streptococcal vaccine.

Adolescent↗
PubMed1994

Comparison of conservation within and between the Ser/Thr and Tyr protein kinase family: proposed model for the catalytic domain of the epidermal growth factor receptor.

The protein kinase family can be subdivided into two main groups based on their ability to phosphorylate Ser/Thr or Tyr substrates. In order to understand the basis of this functional difference, we have carried out a comparative analysis of sequence conservation within and between the Ser/Thr and Tyr protein kinases. A multiple sequence alignment of 86 protein kinase sequences was generated. For each position in the alignment we have computed the conservation of residue type in the Ser/Thr, in the Tyr and in both of the kinase subfamilies. To understand the structural and/or functional basis for the conservation, we have mapped these conservation properties onto the backbone of the recently determined structure of the cAMP-dependent Ser/Thr kinase. The results show that the kinase structure can be roughly segregated, based upon conservation, into three zones. The inner zone contains residues highly conserved in all the kinase family and describes the hydrophobic core of the enzyme together with residues essential for substrate and ATP binding and catalysis. The outer zone contains residues highly variable in all kinases and represents the solvent-exposed surface of the protein. The third zone is comprised of residues conserved in either the Ser/Thr or Tyr kinases or in both, but which are not conserved between them. These are sandwiched between the hydrophobic core and the solvent-exposed surface. In addition to analyzing overall conservation in the kinase family, we have also looked at conservation of its substrate and ATP binding sites. The ATP site is highly conserved throughout the kinases, whereas the substrate binding site is more variable. The active site contains several positions which differ between the Ser/Thr and Tyr kinases and may be responsible for discriminating between hydroxyl bearing side chains. Using this information we propose a model for Tyr substrate binding to the catalytic domain of the epidermal growth factor receptor (EGFR).

Amino Acid Sequence↗
PubMed2006

Single-channel analysis of a point mutation of a conserved serine residue in the S2 ligand-binding domain of the NR2A NMDA receptor subunit.

We have examined the function of a conserved serine residue (Ser670) in the S2 ligand-binding region of the NR2A N-methyl-d-aspartate (NMDA) receptor subunit, using recombinant NR1/NR2A receptors expressed in Xenopus laevis oocytes. Mutation of Ser670 to glycine (S670G) in NR2A reduced the potency of glutamate by 124-fold. Single-channel conductance and the duration of apparent open periods of NR2A(S670G) receptor mutants were, however, indistinguishable from wild-type NMDA receptors. NR1/NR2A(S670G) shut-time distributions were best described by a mixture of six exponential components, and the four shortest shut intervals of each distribution were considered to occur within a channel activation (burst). Bursts of single-channel openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer and had mean durations of 9.6 +/- 0.5 and 29.6 +/- 1.5 ms. The overall channel open probability during a burst was high (mean, 0.83 +/- 0.06). Consistent with a shortening of NMDA receptor-channel burst lengths was the observation of an increased deactivation rate of macroscopic currents evoked by brief applications of glutamate to outside-out membrane patches. Correlations between shut times and adjacent open times were observed in all data records. Noticeably, shorter than average openings tended to occur next to long closed periods, whereas longer than average openings tended to occur next to short closings. Our single-channel data, together with modelling using a kinetic scheme to describe channel activations, support our hypothesis that the S670G point mutation reduces the dwell time of glutamate in its binding site.

Animals↗
PubMed2000

The TetA(K) tetracycline/H(+) antiporter from Staphylococcus aureus: mutagenesis and functional analysis of motif C.

Conserved motif C, identified within members of the major facilitator superfamily (MFS) of transport proteins that mediate drug export, was examined in the tetracycline resistance efflux protein TetA(K) from Staphylococcus aureus; motif C is contained within transmembrane segment 5. Using site-directed mutagenesis, the importance of the conserved glycine (G151, G155, G159, and G160) and proline (P156) residues within this motif was investigated. Over 40 individual amino acid replacements were introduced; however, only alanine and serine substitutions for glycine at G151, G155, and G160 were found to retain significant levels of tetracycline resistance and transport activity in cells expressing mutant proteins. Notably, P156 and G159 appear to be crucial, as amino acid replacements at these positions either significantly reduced or abolished tetracycline/H(+) activity. The highly conserved nature of motif C and its distribution throughout drug exporters imply that the residues of motif C play a similar role in all MFS proteins that function as antiporters.

Amino Acid Motifs↗
PubMed2002

Expression and genomic analysis of midasin, a novel and highly conserved AAA protein distantly related to dynein.

BACKGROUND: The largest open reading frame in the Saccharomyces genome encodes midasin (MDN1p, YLR106p), an AAA ATPase of 560 kDa that is essential for cell viability. Orthologs of midasin have been identified in the genome projects for Drosophila, Arabidopsis, and Schizosaccharomyces pombe. RESULTS: Midasin is present as a single-copy gene encoding a well-conserved protein of approximately 600 kDa in all eukaryotes for which data are available. In humans, the gene maps to 6q15 and encodes a predicted protein of 5596 residues (632 kDa). Sequence alignments of midasin from humans, yeast, Giardia and Encephalitozoon indicate that its domain structure comprises an N-terminal domain (35 kDa), followed by an AAA domain containing six tandem AAA protomers (approximately 30 kDa each), a linker domain (260 kDa), an acidic domain (approximately 70 kDa) containing 35-40% aspartate and glutamate, and a carboxy-terminal M-domain (30 kDa) that possesses MIDAS sequence motifs and is homologous to the I-domain of integrins. Expression of hemagglutamin-tagged midasin in yeast demonstrates a polypeptide of the anticipated size that is localized principally in the nucleus. CONCLUSIONS: The highly conserved structure of midasin in eukaryotes, taken in conjunction with its nuclear localization in yeast, suggests that midasin may function as a nuclear chaperone and be involved in the assembly/disassembly of macromolecular complexes in the nucleus. The AAA domain of midasin is evolutionarily related to that of dynein, but it appears to lack a microtubule-binding site.

Journal Article↗
PubMed1991

Sequence analysis of firefly luciferase family reveals a conservative sequence motif.

A conservative sequence motif was extracted from an alignment of the firefly luciferase family. AngR derived from a pathogenic bacterium and acetyl-CoA synthetases derived from two ascomycete fungi were identified as members of the firefly luciferase family by a homology search with the motif and other sequence comparison analyses. The motif sequence shares several characteristics with the phosphate-binding sites of phosphoproteins and nucleotide-binding proteins. A multiple alignment and an unrooted phylogenetic tree were constructed for the investigation of evolutionary relationships within the firefly luciferase family.

Amino Acid Sequence↗