Modulation of Nr-13 antideath activity by peptide aptamers.

Tumor cells are characterized by deregulated proliferation and resistance to proapoptotic stimuli. The Bcl-2 family of antiapoptotic proteins is overexpressed in a large number of chemoresistant tumors. Downregulation or inhibition of antiapoptotic proteins might result in the sensitization of cancer cells to chemotherapeutic agents. In the present study, we took advantage of the peptide aptamer strategy to target Nr-13, a Bcl-2 antiapoptotic protein involved in neoplastic transformation by the Rous sarcoma virus. We isolated peptide aptamers that behave as Nr-13 regulators, in vitro and in mammalian cells in culture. Some of these aptamers have potential proapoptotic activities. These data suggest that peptide aptamers targeting the Bcl-2 family of apoptosis inhibitors may be useful for the development of anticancer molecules.

Detection of allelic losses on 17q12-q21 chromosomal region in benign lesions and malignant tumors occurring in a familial context.

A predisposing gene (BRCA-1) for breast and ovarian cancer has been located on chromosomal region 17q12-21. According to Knudson's hypothesis if this gene is a tumor suppressor gene, allelic losses would be found in tumors occurring in families with cancer aggregations. We studied 25 samples of both benign lesions and malignant tumors, from breast cancer site-specific families and other familial cancer aggregations. Allelic losses seem to be more frequent in tumors from breast site-specific families but also include the predisposing locus in other syndromes, suggesting a role of BRCA-1 in such families. Finding of allele losses near this locus in benign lesions suggests that such alterations may represent a first step in breast carcinogenesis. It is noteworthy that allele losses involve larger chromosome fragments in malignant tumors than in benign lesions where BRCA-1 is not lost, suggesting a similar mechanism for genomic deletion in the tumorigenesis of the colon and of the breast.

Nrh, a human homologue of Nr-13 associates with Bcl-Xs and is an inhibitor of apoptosis.

In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans. Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes. The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2. RT-PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys. Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end. Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized. Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope. Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.

Two germ-line mutations affecting the same nucleotide at codon 257 of p53 gene, a rare site for mutations.

Codon 257 of the p53 gene is an extremely rare target for somatic mutations (accounting for only two of 1600 published mutations). We report here two constitutional mutations both affecting the second nucleotide of codon 257. A thymine to adenine transversion resulting in an amino acid change from leucine to glutamine was found in one proband who developed multiple independent malignant tumors (osteosarcoma, phyllodes tumor, soft-tissue sarcoma). Her mother died of early-onset breast cancer. In the other case, a deletion resulting in a frameshift in the C-terminal coding region of p53 was found in a woman who was diagnosed with breast cancer at age 34. This woman belongs to a family with features of Li-Fraumeni syndrome. In both cases, the p53 mutations identified in the proband was found in other members of the family. Codon 257, even if rarely mutated in somatic cells, may thus be an important target for germ-line mutations.

Genomic stability and wild-type p53 function of lymphoblastoid cells with germ-line p53 mutation.

Increased cancer risk associated with germ-line p53 mutation was linked to a deficit in the ability to maintain genomic stability. Accordingly, normal fibroblasts from cancer-prone individuals accumulate genomic aberrations with concomitant loss of wild-type p53 allele during in vitro culture. We tested whether such changes also occur in EBV-immortalized lymphoblastoid cells. Both normal and p53 germ-line mutant lymphoblastoid cells maintained functional p53 and genomic stability during long term in vitro culture. These unexpected differences between fibroblastic and lymphoblastic cells suggest that phenotypic expression of p53 deficiency is cell type specific. This could contribute to selective tissular localization of tumours observed in patients with Li-Fraumeni syndrome despite the presence of a mutant p53 allele in all cells.

Lack of germ-line mutations in the p53 gene exons 4 to 8 in patients with late-onset second malignant neoplasms.

Previous studies have shown p53 germ-line mutations in some familial cancer aggregations with or without the Li-Fraumeni syndrome (LFS). Such mutations were also reported in children and young adults with second malignant neoplasms (SMN). This led us to screen for p53 germ-line mutations in a group of seven patients affected with SMN, but characterized by an older age of onset than in the previous reports. No mutation was found in exons 4 to 8 and their boundaries using the single-strand conformation polymorphism technique. Our results give strong evidence for genetic heterogeneity of SMN, probably related to the age of cancer onset.

[Hereditary predisposition for cancer of the breast and the ovary]. / Prédisposition héréditaire aux cancers du sein et de l'ovaire.

Familial breast cancers represent about 10% of all cases of breast cancers. A predisposition locus has been located in the 17q21 chromosomal region. Nineteen French breast and breast-ovarian cancer families were tested for linkage with five highly polymorphic 17q markers. The five breast-ovarian cancer families as a group give positive evidence for linkage, whereas the 14 breast cancer families do not. Heterogeneity of linkage of familial breast cancer is significant in France and supports the existence of more than one susceptibility gene.

Veterans Affairs Multicenter Low Vision Enhancement System (LVES) study: clinical results. Report 1: effects of manual-focus LVES on visual acuity and contrast sensitivity.

PURPOSE: The Department of Veterans Affairs LVES Study is a multicenter study to determine the effectiveness of the Low Vision Enhancement System (LVES) as a visual rehabilitation device. The purpose of this study was to explore the efficacy of the Beta 1 manual-focus LVES for improving visual acuity and contrast sensitivity. METHODS: Patients whose visual acuity was 20/80 or worse in the better eye from any disease, who did not have significant visual field loss, who had previous low vision experience and were capable of working with the LVES were enrolled in a comprehensive prospective multicenter clinical evaluation. Initially, corrected spectacle visual acuities were measured using a standardized ETDRS chart. Contrast sensitivities were also measured with spectacle correction using a standardized Peli-Robson chart. These results were then compared to the acuities and contrast sensitivity obtained with the LVES at optimal magnification. Also, visual acuities were measured using an Eschenbach 3x spectacle-mounted binocular telescope, then compared to the acuities obtained using the LVES set at the lowest magnification (3x). RESULTS: All patients who completed the study demonstrated an improvement in visual acuity, with a median improvement of six lines of Snellen equivalent acuity using the LVES. Improvement in visual acuity was the same in both ARMD and non-ARMD causes of vision loss. Mean contrast sensitivity improved in 52 of 58 patients tested, with a mean improvement of 0.49 log units. CONCLUSION: The LVES significantly improves both visual acuity and contrast sensitivity in visually impaired patients who fall within the study criteria. Up to 10-fold improvement in visual acuity and up to 1.80 log units improvement in contrast sensitivity were noted in the study group when the LVES was used.

Central retinal vein occlusion and iris neovascularization hemorrhage.

Although there have been few direct observations, the etiology of spontaneous hyphema in patients with retinal or ocular hypoxia is assumed to be hemorrhage from a neovascular iris vessel. This paper reports observed hemorrhage from such a rubeotic iris in a patient with central retinal vein occlusion, diabetes, hypertension, peripheral vascular disease and chronic open-angle glaucoma. Bleeding was spontaneous with dilation, but stopped within 24 hours without treatment, leaving only traces of inferior angle blood staining. The two types of central retinal vein occlusion, and suggestions for their management, are also discussed.

How reliable is the Goldmann tonometer as a standard?

In a clinical comparison of an XPERT NCT non-contact tonometer with Goldmann applanation tonometry (GAT), reported in the companion article, we concluded that the XPERT is a highly accurate and reliable instrument. By design, our evaluation differed significantly from earlier clinical studies in its use of multiple references (four calibrated GAT reference tonometers and seven GAT operators). Five clinicians were responsible for GAT measurement of 400 of a 620 eye population. Each clinician's population sample was essentially drawn at random from the common pool. XPERT vs. GAT performance indices were calculated for each of the five GAT operators. Because XPERT's measurements are essentially free of operator influence, it is reasonable to conclude that differences found among these five "XPERT performance" findings were primarily attributable to differences in GAT operator technique and endpoint judgment. Our findings disclose compelling evidence that GAT's accuracy and reliability depends on individual operator technique and experience. They indicate that in a clinical comparison, the protocol safeguards proposed by Wittenberg need be applied to ensure the integrity of studies using GAT as the reference tonometer.

Automated perimetry in the management of glaucoma.

As optometry's role and responsibilities increase, so does our need to understand and utilize new technological advances to aid in our understanding and management of ocular conditions. Automated perimetry represents one such technological improvement that enhances our ability to detect and analyze the visual field changes associated with glaucoma. An understanding of the automated perimeter's abilities and limitations is needed to effectively incorporate it into the management of glaucoma.

XPERT NCT--a clinical evaluation.

A clinical evaluation of the XPERT NCT (Reichert Ophthalmic Instruments) applanation non-contact tonometer was conducted at the Baltimore VA Medical Center. Each of 620 eyes were measured by the XPERT and then by one of seven operators of varied experience, each using one of four calibrated Goldmann applanation tonometers (GATs). Measured pressures ranged from 1 to more than 60 mmHg. The interval between XPERT and GAT measurements varied from a few minutes to almost 1 hour. Despite the increased potential variability of our protocol, the XPERT agreed very closely with GAT. Population IOP means agreed within 0.3 mmHg; regression coefficients defining XPERT's calibration, relative to GAT, were slope (m) = 1.025, and Y intercept (b) = 0.7 mmHg. The standard deviation of matched pair differences, was Sd = 2.7 mmHg. We conclude that the XPERT NCT is a highly accurate and reliable instrument. Our experience showed it to be convenient and well accepted by staff and patients. It proved faster, easier to use, and its "air-puff" is quieter and less intense than NCT I and II. In the companion paper, we examined the reliability of GAT used as a clinical comparison reference in evaluating the XPERT.

A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: implications for a second XPG function.

Xeroderma pigmentosum (XP) patients have defects in nucleotide excision repair (NER), the versatile repair pathway that removes UV-induced damage and other bulky DNA adducts. Patients with Cockayne syndrome (CS), another rare sun-sensitive disorder, are specifically defective in the preferential removal of damage from the transcribed strand of active genes, a process known as transcription-coupled repair. These two disorders are usually clinically and genetically distinct, but complementation analyses have assigned a few CS patients to the rare XP groups B, D, or G. The XPG gene encodes a structure-specific endonuclease that nicks damaged DNA 3' to the lesion during NER. Here we show that three XPG/CS patients had mutations that would produce severely truncated XPG proteins. In contrast, two sibling XPG patients without CS are able to make full-length XPG, but with a missense mutation that inactivates its function in NER. These results suggest that XPG/CS mutations abolish interactions required for a second important XPG function and that it is the loss of this second function that leads to the CS clinical phenotype.

Conserved residues of human XPG protein important for nuclease activity and function in nucleotide excision repair.

The human XPG endonuclease cuts on the 3' side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3' cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.

High allele loss rates at 17q12-q21 in breast and ovarian tumors from BRCAl-linked families. The Breast Cancer Linkage Consortium.

Loss of heterozygosity (LOH) was evaluated in 174 breast and ovarian tumors derived from 94 families with at least 3 first-degree relatives affected with either of these cancers. By linkage analysis 26 families were identified as having a high posterior probability of being due to BRCAl (the breast/ovarian cancer susceptibility locus on 17q12-21) with lod scores varying from 0.51 to 9.49. Tumor genotypes were determined at at least 2 constitutionally heterozygous markers flanking BRCAl in a total of 58 tumors from these families. These tumors were derived from 52 patients, the BRCAl mutation carrier status of which was evidenced by DNA sequencing in 20, and inferred by reconstructing haplotypes in the remainder. LOH was detected in 50 (86%) tumors, and invariably involved the wild-type allele. Where informative, this allele was of paternal origin in 33 cases and of maternal origin in 10 cases. These results strongly suggest that BRCAl is a tumor suppressor gene and that LOH is greatly favored to fully inactivate it.

Linkage analysis of 19 French breast cancer families, with five chromosome 17q markers.

Nineteen French breast and breast-ovarian cancer families were tested for linkage with five chromosome 17q markers. The five breast-ovarian cancer families as a group give positive evidence for linkage, whereas the 14 breast cancer-only families do not. Heterogeneity of linkage of breast and breast-ovarian cancers is significant in France and supports the existence of more than one susceptibility gene.