Luminosity-independent measurement of the proton-proton total cross section at √s=8 TeV.

The TOTEM collaboration has measured the proton-proton total cross section at √s=8 TeV using a luminosity-independent method. In LHC fills with dedicated beam optics, the Roman pots have been inserted very close to the beam allowing the detection of ~90% of the nuclear elastic scattering events. Simultaneously the inelastic scattering rate has been measured by the T1 and T2 telescopes. By applying the optical theorem, the total proton-proton cross section of (101.7±2.9) mb has been determined, well in agreement with the extrapolation from lower energies. This method also allows one to derive the luminosity-independent elastic and inelastic cross sections: σ(el)=(27.1±1.4) mb; σ(inel)=(74.7±1.7) mb.

Double diffractive cross-section measurement in the forward region at the LHC.

The first double diffractive cross-section measurement in the very forward region has been carried out by the TOTEM experiment at the LHC with a center-of-mass energy of sqrt[s]=7 TeV. By utilizing the very forward TOTEM tracking detectors T1 and T2, which extend up to |η|=6.5, a clean sample of double diffractive pp events was extracted. From these events, we determined the cross section σDD=(116±25) µb for events where both diffractive systems have 4.7<|η|min<6.5.

Design of a radiation surveillance unit for an unmanned aerial vehicle.

This paper describes a prototype of a compact environmental radiation surveillance instrument designed for a Ranger unmanned aerial vehicle. The instrument, which can be used for tracking a radioactive plume, mapping fallout and searching for point sources, consists of three different detector types (GM, NaI(Tl) and CZT) and an air sampling unit. In addition to the standard electronics for data acquisition, the system contains an onboard computer, a GPS receiver and environmental sensors, all enclosed in a single housing manufactured of fiberglass-reinforced composite material. The data collected during the flight is transmitted in real-time to the ground station via a TETRA radio network. The radiation surveillance unit is an independent module and as such can be used in, for example, airplanes, helicopters and cars.

Variants of the long control region of human papillomavirus type 16.

Expression of the human papillomavirus (HPV) E6 and E7 oncogenes is regulated on the transcriptional level by specific protein-binding sites contained in the viral long control region (LCR). Sequence changes within the LCR region may have an impact on the transcription of viral oncogenes, possibly resulting in differences in the oncogenic potential of the virus. The present study was designed to determine the sequence variability of the LCR of HPV 16 and to assess whether certain LCR variants do correlate with the clinical outcome of the disease of the uterine cervix. The entire LCR segment of HPV 16 was analysed from 37 cervical biopsy specimens derived from 28 women included in the Kuopio long-term prospective follow-up study. The LCR sequence was identical with the reference sequence in six HPV 16 isolates. Overall, 14 different HPV 16 LCR variants were identified. One of the variants showed sequence variation typical of the Asian-American variant lineage of HPV 16, and all the other variants appeared to belong to the European variant group. The European variants exhibited low genetic diversity, and only five of these LCR variants contained nucleotide changes involving known or proposed binding sites for transcription factors. The variants with changes at nucleotide positions 7193 and 7521 was the most prevalent, accounting for almost 37% of infections. This variant (7193; 7521) has been previously demonstrated to have similar transcriptional activity compared with the reference isolate by Veress and colleagues J Gen Virol 1999, 80, 1035-1043. The reference isolate, variant (7193; 7521) and variants with changes within transcription factor binding sites accounted for most of the infections, and no significant differences were found in the comparison of the distribution of these different LCR isolates in cases where the disease showed progression to severe cervical intraepithelial neoplasia (CIN) or carcinoma in situ (CIS). Notably, both the reference isolate and variant (7193; 7521) were also closely associated with infections showing more aggressive behaviour. According to the present findings, in European HPV 16 isolates, intratype genetic variation of the LCR region does not seem to be commonly responsible for differences in the pathogenicity of the virus and thereby for a risk of progressive infections.

p53 mutations and presence of HPV DNA do not correlate with radiosensitivity of gynecological cancer cell lines.

OBJECTIVE: The correlation between p53 tumor suppressor gene mutations and the presence of high-risk human papillomavirus (HPV) DNA with the in vitro radiosensitivity of gynecological malignancies was studied in 26 cell lines derived from gynecological cancers of 23 patients. METHODS: Comparison of the intrinsic radiosensitivity was performed with mean inactivation dose (D) determined with the 96-well plate clonogenic assay. p53 mutations were investigated with polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis and direct DNA sequencing, and the presence of HPV DNA was studied with PCR using HPV consensus primers. RESULTS: p53 mutations were found in 6 of 10 vulvar squamous cell carcinoma (SCC) lines. Nine vulvar and 1 vaginal SCC cell lines were HPV DNA negative and 1 vulvar cell line was HPV 16 positive. All 4 cervical SCC lines were HPV positive and possessed the wild-type p53. Three cell lines expressed HPV 16 and 1 HPV 68. Among 10 endometrial cancer cell lines, 2 cell lines with mutant p53 and 1 HPV 16 positive cell line were found. No correlation could be demonstrated between inactivation of the p53 gene and radiosensitivity in vitro; the cell lines were evaluated as one group or according to their anatomical origin or histology. CONCLUSION: Our results indicate that inactivation of the p53 gene through mutation or binding with HPV DNA does not increase the resistance of gynecological malignancies to ionizing radiation in vitro.

p53 and bcl-2 proteins as prognostic markers in human papillomavirus-associated cervical lesions.

PURPOSE: The present study was designed to analyze the expression of p53, mdm2, and bcl-2 proteins, with special emphasis on their association with the grade of squamous intraepithelial lesion (SIL), human papillomavirus (HPV) type, and clinical course of the disease. Special attention was focused on the value of individual protein expressions, as well as combined p53/mdm2 and p53/bcl-2 phenotypes, in predicting the clinical course of cervical lesions. MATERIALS AND METHODS: The expression of p53, mdm2, and bcl-2 was studied immunohistochemically in a series of 98 HPV lesions of the uterine cervix. RESULTS: Frequent expression of p53, mdm2, and bcl-2 proteins was found in the cervical lesions. However, only p53 expression independently provided information for prediction of the clinical course of HPV lesions. High levels of p53 expression were detected in most low-grade SILs (LSILs) (83%) and HPV 6/11/42-associated lesions (86%). In high-grade SILs (HSILs) positive for high-risk HPV types, p53 expression was frequently either totally absent or it only occurred in a few scattered cells. These lesions were closely associated with disease progression. The evaluation of bcl-2 expression alone was not useful for predicting clinical outcome, although abnormal bcl-2 expression in suprabasal layers was more common in HSILs. By contrast, the combined p53/bcl-2 phenotype, which showed a low percentage of p53-positive cells with bcl-2 overexpression in upper epithelial layers, was found to be involved in the progression of HPV lesions. CONCLUSION: The present study showed that HPV lesions with a high percentage of cells that express p53 are more likely to regress than those with low or absent p53. p53 thus seems to hold promise as a molecular marker for the risk of the progression of HPV-associated SILs. In addition, the assessment of p53 and bcl-2 expression patterns may be useful to predict the clinical course of cervical HPV lesions.

SSCP pattern indicative for p53 mutation is related to advanced stage and high-grade of tongue cancer.

53 and bcl-2 are involved in the control of cell cycling and apoptosis. Environmental factors such as smoking and radiation can disturb p53 function and predispose a cell to malignant transformation. To investigate the role of p53 mutations, as well as p53 and bcl-2 protein expression in squamous cell carcinoma of the tongue, 39 samples were analysed. Since neck metastasis is the most important prognostic factor of this disease, samples from patients both with and without nodal disease were selected to find out whether there was any difference between the groups. Non-radioactive single-stranded conformation polymorphism (SSCP) was used to screen p53 mutations; an altered SSCP pattern indicating p53 mutation was found in 21 samples (54%). A significant correlation between tumour size, histological differentiation and p53 mutations was found (P < 0.01). Immunocytochemically, nuclear expression of p53 was moderate or strong in 18 (46%) samples. No correlation between altered p53 SSCP pattern and p53 immunoreactivity was seen. bcl-2 expression was cytoplasmic; moderate or strong staining was detected in only six of the carcinoma samples (15.5%). Interestingly, there was a significant correlation between smoking and bcl-2 expression (P < 0.01): all six samples with moderate or strong staining were taken from heavy smokers. Furthermore, all those patients died within 32 months.

Mutation of tumor suppressor gene p53 is frequently found in vulvar carcinoma cells.

OBJECTIVE: The purpose of this study was to evaluate the presence and type of mutations of the tumor suppressor gene p53 in squamous carcinoma cell lines of the vulva. STUDY DESIGN: Eight low-passage cell lines established from vulvar carcinoma were included in the analysis. Mutational analysis was restricted to exons 5 through 9 of the p53 gene, previously shown to have a high incidence of mutations. The sequences containing exons 5/6,7, and 8/9 were amplified by polymerase chain reaction and screened with a single-strand conformation polymorphism technique on PhastSystem (Pharmacia Biotech, Uppsala, Sweden). Exons from samples showing mobility shifts in single-strand conformation polymorphism were sequenced by polymerase chain reaction direct sequencing. RESULTS: Five vulvar carcinoma cell lines showed abnormal electrophoretic mobility of exons 5/6, one of exons 8/9, and one of exon 7. Reduction to homozygosity was detected in four vulvar carcinoma cell lines. Missense mutations were detected by sequence analysis in UM-SCV-2 (codon 171: GAG[Glu]-->TAG[STOP]), UM-SCV-3 (hot spot codon 273: CGT[Arg]-->TGT[Cys]), UM-SCV-4 (codon 151: CCC[Pro]-->CAC[His]), UM-SCV-5 (codon 155: ACC[Thr]-->ATC[lle]), and UM-SCV-7 (codon 245: GGC[Gly]-->AGC[Ser]). UM-SCV-3 also carried a missense mutation with no amino acid change (codon 314: TCC[Ser]-->TCT[Ser]). UM-SCV-7 carried an additional base deletion at codon 249 (AGG-->AG-), likely resulting in a frameshift in transcription and a truncated protein product. Four of the seven mutations were transitions, two were transversions, and one was a deletion. The presence of transitions suggests that at least a proportion of p53 mutations of these cancers may arise spontaneously without exogenous carcinogen exposure. UM-SCV-1A and UM-SCV-1B were derived from the primary tumor and pleural effusion of the same patient. UM-SCV-6 is a cell line that contains human papillomavirus 16. No mutations in these three cell lines were found by single-strand conformation polymorphism. CONCLUSIONS: On the basis of previous observations, loss of tumor suppressor p53 function either by mutation or human papillomavirus involvement is a frequent phenomenon in cervical carcinoma cells. It appears now that functional inactivation of p53 is associated also with vulvar carcinoma cell lines, but mutations of the p53 gene are much more common in vulvar than in cervical carcinoma cell lines.

p53 protein detected by immunohistochemistry as a prognostic factor in patients with epithelial ovarian carcinoma.

BACKGROUND: The clinical significance of p53 suppressor gene nucleoprotein immunostaining in ovarian epithelial cancer has not been determined. METHODS: p53 protein expression was studied by immunohistochemistry from paraffin embedded tissue in a series of 136 patients with malignant ovarian epithelial tumors. The median follow-up time of the patients still alive was 10 years. RESULTS: Sixty (44%) carcinomas stained clearly positive for p53 protein. Positive staining for p53 protein was associated with the serous histologic type (P = 0.0006), a higher than the median S-phase fraction size determined by DNA flow cytometry (P = 0.02), and poor histologic grade of differentiation (P = 0.04), but not with the International Federation of Gynecology and Obstetrics (FIGO) stage, age at diagnosis, or DNA ploidy. Cancers with positive staining had only 17% 5-year and 9% 15-year survival rates compared with 42% 5-year and 36% 15-year survival rates corrected for intercurrent deaths among the rest of patients (P = 0.002). In a multivariate analysis, positive p53 staining was associated with poor survival (relative risk of death, 1.8, 95% confidence interval [CI], 1.2-2.9) together with less than radical surgery (nonradical vs. radical: RR, 5.5; 95% CI, 2.2-13.6), and advanced FIGO stage (RR, 1.4; 95% CI, 1.0-2.0). CONCLUSION: Although p53 protein immunostaining is associated with several other prognostic factors in epithelial ovarian cancer, it may also have independent prognostic value in this disease.

Rapid and effective detection of mutations in the p53 gene using nonradioactive single-strand conformation polymorphism (SSCP) technique applied on PhastSystem.

The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method is a powerful tool for the screening of genetic alterations, including single-base substitutions. In the present study, the conventional SSCP technique was modified on the semiautomated electrophoresis system (PhastSystem) for the detection of mutations in the p53 tumor suppressor gene. The SSCP running conditions were optimized for three PCR-amplified DNA fragments, spanning exons 5 through 9 of the p53 gene, using the PCR-products derived from the CaSki and HaCaT cells as the normal and mutant controls, respectively. The optimized SSCP protocols were tested on nine human vulvar and vaginal carcinoma-derived cell lines. The optimizing experiments indicated that the running temperature and gel density can affect significantly the electrophoretic mobility and resolution of single-stranded DNA molecules. Because the gel temperature is the most important parameter affecting the conformation and thus electrophoretic mobility of single strands, one of the most important advantages of the SSCP technique on the PhastSystem is that the running temperature is controlled precisely. In addition to the fast electrophoretic separation, the PhastSystem also offers the use of a silver staining method allowing direct visualization of DNA with high detection sensitivity. Thus, the important advantage of this modified SSCP technique is the short time required for analysis, including electrophoresis and DNA detection. It is concluded that the SSCP method applied on the PhastSystem has the advantages of simplicity, efficiency, speed and reproducibility, and is suitable for clinical diagnostic purposes.

Frequent mutations of p53 gene in oesophageal squamous cell carcinomas with and without human papillomavirus (HPV) involvement suggest the dominant role of environmental carcinogens in oesophageal carcinogenesis.

Epidemiological evidence suggests that alcohol intake, use of tobacco, ingestion of mycotoxins and nitrosamines and nutritional deficiencies are high-risk factors for the development of oesophageal cancer. Similarly, viral infections have been postulated to play a role in some tumours. However, the molecular events underlying the development of oesophageal carcinoma are poorly understood as yet. Loss of p53 tumour-suppressor gene function has been found in different human malignancies, and it can occur in a variety of ways, including gene mutation and interaction with the E6 protein of oncogenic human papillomaviruses (HPVs). Because the oesophageal mucosa is potentially exposed to mutagens and HPVs, we studied DNA samples derived from nine HPV-positive squamous cell carcinomas and 12 HPV-negative tumours. Exons 5-9 of the p53 gene containing phylogenetically conserved domains were examined using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique. HPV detection was done using DNA in situ hybridisation with biotin-labelled HPV DNA probes. Mutations were detected in eight (38%) out of the 21 cases. Three mutations were found in exons 5/6, three in exon 7 and two in exon 8/9. Six (50%) of the 12 HPV-negative carcinomas showed p53 mutations. Two (22.2%) of the nine HPV-positive carcinomas were found to contain p53 mutations as well; one contained HPV 16 DNA sequences and showed p53 mutation in exon 8/9, and the other was HPV 6/11 positive with the mutation in exon 5/6. Although mutations were more common in HPV-negative tumours (50.0% vs 22.2%), the difference in p53 mutations in HPV-positive and -negative tumours did not reach statistical significance (P = 0.1946). These data indicate that inactivation of the p53 gene is a frequent event in oesophageal squamous cell carcinomas and such an inactivation might be an important molecular pathway for the development of oesophageal cancer. The findings of p53 mutations in HPV-positive oesophageal carcinomas suggest that HPV and p53 mutation were not mutually exclusive events. The presence of frequent mutations of p53 gene in both HPV-positive and -negative oesophageal carcinomas suggests a dominant role of environmental carcinogens in oesophageal carcinogenesis.

Testing for mutations in exon 17 of the beta-amyloid precursor protein gene in Finnish Alzheimer patients and normal subjects.

Mutations in the beta-amyloid precursor protein gene on chromosome 21 were shown to cause a small proportion of Alzheimer's disease. We studied the occurrence of the point mutations in exon 17 of the beta-amyloid precursor protein gene in a sample of Finnish familial Alzheimer patients and nondemented controls using polymerase chain reaction and a single strand conformation polymorphism technique. In addition, mutations in familial Alzheimer's disease patients were studied by sequencing the amplified products. Interestingly, two probable polymerase chain reaction errors were detected in codons 717 and 693 of the exon 17. However, no mutations in the exon 17 were confirmed adding the study to the body of literature that mutations in the exon 17 are a rare cause of familial Alzheimer's disease.

The state of the p53 gene in human papillomavirus (HPV)-positive and HPV-negative genital precancer lesions and carcinomas as determined by single-strand conformation polymorphism analysis and sequencing.

Human papillomavirus (HPV) is frequently associated with cervical carcinoma. Inactivation of the p53 tumor suppressor gene product by binding to the HPV encoded E6 protein is considered as an important pathway for malignant progress in HPV-infected cells. In contrast, mutations of the p53 gene have been found in HPV-negative cervical carcinoma cells. To evaluate the involvement of p53 inactivation for the development of genital carcinoma, we determined the state of the p53 gene in 20 genital precancer lesions and carcinomas, which had been previously studied for the expression of p53 protein and the presence of HPV DNA. Exons 5 through 9 of the p53 gene were analyzed by single-strand conformation polymorphism analysis of polymerase chain reaction (PCR)-amplified DNA fragments, and the results obtained by the PCR-SSCP analysis were confirmed by DNA sequencing. No mutations were detected in any of the specimens, including the three HPV-negative cases. The present results suggest that the functional inactivation of p53 is not invariably required for the induction of malignant transformation in the genital tract, and thus other genetic events can also significantly participate in genital carcinogenesis.

The p53 tumor suppressor gene as a common cellular target in human carcinogenesis.

The p53 gene is a 16-20 kb of cellular DNA located on the short arm of human chromosome 17 at position 17p13.1. This gene encodes a 375-amino acid nuclear phosphoprotein which involves in the regulation of cell proliferation. The p53 gene was originally regarded as a dominant oncogene because its overexpression resulted in the immortalization of rodent cells, and the p53 gene could transform rat embryonic fibroblasts in concert with an activated ras gene. It soon became clear, however, that many of the p53 clones that had been studied were in fact mutated versions of the gene, and the wild-type p53 actually acts as a tumor suppressor. Loss of normal p53 function has been associated with the cell transformation in vitro and the development of neoplasms in vivo. More than one-half of human malignancies derived from the epithelial, mesenchymal, hematopoietic, and lymphoid tissues, as well as the central nervous system, analyzed thus far, were shown to contain an altered p53 gene. Most p53 gene alterations are the missense mutations, giving rise to an altered protein. These mutations are most frequently located in the evolutionally conserved areas. Furthermore, it has been demonstrated that the SV40 large T antigen, the adenovirus E1B protein, and papillomavirus E6 protein can bind to wild-type p53 protein and presumably lead to inactivation of this gene product as well. Therefore, the inactivation of normal (or wild-type) p53 is currently regarded as an important genetic pathway for human carcinogenesis generated by endogenous factors and exogenous carcinogens, as well as several tumor viruses. The current data on the p53 gene and its alterations in human malignancies, particularly those in the gastrointestinal tract, are reviewed.