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Determination of aflatoxin B1 (AFB1) in wheat has been accomplished by enhanced spectrofluorimetry in combination with second-order standard addition method (EF-SOSAM). The adopted strategy combined the use of parallel factor analysis (PARAFAC) for extraction of the pure analyte signal and the standard addition method, for a determination in the presence of matrix effect caused by wheat matrix. The method is based on the enhanced fluorescence of AFB1 by beta-cyclodextrin in 10% (w/w) methanol-water solution. After sample treatment and without any extended cleanup steps and derivatization process, four standard additions were performed for each sample. A specific PARAFAC model was built from three-way arrays formed by excitation-emission spectra and 5 measurements (sample plus 4 additions). The scores related to AFB1 were used for a linear regression in the standard addition method. Two naturally contaminated wheat and spiked wheat samples containing AFB1 in the range 0-18 microg kg(-1) were analyzed by EF-SOSAM and compared with HPLC results. EF-SOSAM analysis of spiked wheat samples gave a good correlation with spiked values (R(2)>0.990). The limit of detection of method was 0.9 microg kg(-1) for the determination of AFB1 in wheat samples.

In this study we present two novel anaplastic thyroid carcinoma (ATC) lines (HTh 104 and HTh 112) and further characterize six frequently used ATC lines (HTh 7, HTh 74, HTh 83, C 643, KAT-4, and SW 1736). Three of the lines carried a heterozygous BRAF mutation V600E, which is in line with reports of BRAF mutations in primary ATC and papillary thyroid cancer. Several nonrandom breakpoints were identified by spectral karyotyping (SKY) and G-banding in these lines including the novel 1p36 and 17q24-25 as well as 3p21-22 and 15q26 that are also implicated in well-differentiated thyroid cancers. Comparative genomic hybridization showed frequent gain of 20q, including the UBCH10 gene in 20q13.12, which was further confirmed by array-comparative genomic hybridization and fluorescence in situ hybridization analyses. Our results concur with previous studies in both primary tumors and cell lines, indicating that gain of chromosome 20 is important in the pathogenesis of ATC and/or progression of differentiated thyroid cancers to ATC.

The roles of all healthcare professionals have changed considerably over the years and the nurse in particular has been affected. These changes have been influenced by the following guidance, Code of Professional Conduct (UKCC 1992) and Code of Professional Conduct (NMC 2004). Such changes involve the whole area of advancing roles in perioperative nursing, such as the nurse working as advanced scrub practitioner (ASP) (formally known as first assistant). Boss (2002) mentions that these new role developments are concerned with principles such as serving the interests of patients, providing holistic care, developing professional skills and knowledge and being accountable and responsible for your individual actions. Nurses acting as an ASP have many matters to contend with, such as autonomy, duty of care and other legal issues, and professional accountability. This paper explores these issues in more detail.

The determination of aflatoxin B(1) (AFB1) in pistachio has been accomplished by normal and synchronous fluorimetry in combination with some multivariate calibration methods and derivative techniques. Extending the two-dimensional synchronous fluorescence scan to a three-dimensional total synchronous fluorescence scan was used to obtain the optimized Deltalambda for AFB1 in pistachio sample. The methods are based on the enhanced fluorescence of AFB1 by beta-cyclodextrine in 10% (v/v) methanol-water solution. Twenty-six pistachio samples containing AFB1 in the range 0-15 ppb were used as calibration set. Eighteen combinational methods were tested to make best model for prediction of AFB1 and finally best results obtained using a method based on synchronous fluorimetry in combination with multiple linear regressions (MLR). For concentrations ranging from 0 to 15 ppb of AFB1 in 22 pistachio samples as prediction set, the values of root mean square difference (RMSD) and relative error of prediction (REP) using MLR were 0.328 and 4.453%, respectively were observed. Two naturally contaminated pistachio samples were analyzed by synchronous fluorimetry using MLR and compared with HPLC results.

Aim: To determine whether hepatitis C virus (HCV) infection of index cases increases intrafamilial transmission (sexual and nonsexual contacts) of HCV., Methods: In a case-control descriptive study we enrolled 300-household contacts of 60 index cases (40 males and 20 females) of HCV infection and 360 pair-matched controls in Ahwaz JundiShapour University Hospitals from August 1, 1998 to September 1, 2003. The control group consisted of first time blood donors referred to the Regional Blood Transfusion Organization. Serum samples and demographic data and a medical history including the existence of risk factors for HCV (after a questionnaire on the risk factors for parenteral exposure) were obtained from each subject. Antibodies to HCV were detected employing a commercially available second-generation enzyme immunoassay (EIA, Abbott II). Positive serum specimens were retested using a second-generation recombinant immunoblot assay (RIBA-2) and a polymerase chain reaction for HCV RNA. Data analysis was carried out for intra-household clustering., Results: Only 4 of 300 (1.33%) cases of household contacts without percutaneous risk factors were positive for HCV Ab while the remaining 296 family contacts were negative for anti-HCV. The mean age of the index cases was 28.4 (Std 15.22) years. The anti-HCV prevalences in parents, spouses, children of the index cases were 0.87% (1/115), 3.39% (2/59)) and 0.79% (1/126), respectively. Among couple partners negative for anti-HCV antibodies, the mean duration of the sexual relationship was 6 years. The two-couple partners positive for anti-HCV antibodies married the index cases for longer than 15 years. The prevalence of positive HCV Ab among household contacts (1.33%) was not significantly higher than that in the controls (1%) (P > 0.06)., Conclusion: Intrafamilial transmission of HCV is not the significant transmission route and sexual transmission does not seem to play a role in the intrafamilial spread of HCV infection. Intrafamilial transmission of HCV is possible but occurs at a low rate.

Background: Peptic ulceration following infection of the stomach with H. Pylori is a common disease. Accurate and rapid detection of the bacteria can lead to the implementation of appropriate treatment and recovery. Chronic infection of the gastric milieu with H. Pylori may lead to gastric carcinoma. Routine detection of this bacterium in peptic ulcer is based on the urease test and culture of peptic biopsies. Unfortunately, the sensitivity and specificity of both tests are not satisfying. Molecular techniques have been successfully applied for the rapid and accurate detection of bacterial agents in clinical samples. This study was undertaken to evaluate the sensitivity and specificity of fluorescent in situ hybridization (FISH) in the detection of H. Pylori in patients suffering from dyspepsia., Material/methods: One hundred gastric biopsy samples taken by endoscopy from the antrum and corpus of the stomach were tested by FISH and compared with the conventional culture method complemented by biochemical tests., Results: FISH detected H. Pylori in 48 clinical samples, while the conventional method detected 42 samples. The sensitivity and specificity of FISH for the detection of H. Pylori were calculated as 98% and 100%, respectively., Conclusions: The findings of this study suggest that FISH is a highly suitable and rapid method for diagnosing H. Pylori. Especially when the samples are taken from the antrum and the corpus of the stomach, this technique potentially can be applied routinely for the detection of this bacterium in clinical samples.

Aim: To evaluate possible risk factors for the spread of hepatitis C infection and to analyze the characteristics of the epidemiological and clinical patterns among the patients with hepatitis C infection., Methods: During a five-year period a cross-sectional study was conducted among HCV positive individuals referred to the Ahwaz JundiShapour University Hospitals (AJSUH) and Hepatitis Clinic from 1 Sept 1999 to 1 Sept 2003. The control group consisted of first time blood donors referred to the Regional Blood Transfusion organization. Enzyme-linked immunosorbent assay and recombinant immunoblot assay anti-hepatitis C virus (HCV) tests were performed for two groups. Positive serum specimens were retested using polymerase chain reaction (PCR) for HCV RNA. Risk factors were evaluated using a questionnaire. Reported risk factors among infected subjects ("HCV-positive") were compared to those of subjects never exposed ("HCV-negative") to HCV., Results: A total of 514 subjects were studied for HCV, of which 254 were HCV-positive and 260 HCV-negative donors comprised the control group. Mean age of the patients was 28.4 (Std 15.22) years. HCV-positive subjects were more likely to be of male gender (63% versus 37%). Transfusion 132 (52%), non-intravenous (n-i.v.) drug abuse and i.v. drug abuse 37 (14.5%), haemodialysis 25 (10%), receiving wounds at war and extramarital sexual activities (2.4%), tattooing (3.6%) were found to be independent risk factors of being HCV-positive. No apparent risk factors could be demonstrated in 29 (11.2%) of the positive cases., Conclusion: Our data indicate that a history of transfusion and i.v. drug abuse and haemodialysis are important risk factors for HCV infection in our area and that more careful pretransfusion screening of blood for anti-HCV must be introduced in our blood banks. Improvements in certain lifestyle patterns, and customs in this area may be essential to prevent transmission of the infection.

Plastic shear significantly reduces the phase transformation (PT) pressure when compared to hydrostatic conditions. Here, a paradoxical result was obtained: PT of graphitelike hexagonal boron nitride (hBN) to superhard wurtzitic boron nitride under pressure and shear started at about the same pressure ( approximately 10 GPa) as under hydrostatic conditions. In situ x-ray diffraction measurement and modeling of the turbostratic stacking fault concentration (degree of disorder) and PT in hBN were performed. Under hydrostatic pressure, changes in the disorder were negligible. Under a complex compression and shear loading program, a strain-induced disorder was observed and quantitatively characterized. It is found that the strain-induced disorder suppresses PT which compensates the promotion effect of plastic shear. The existence of transformation-induced plasticity (TRIP) was also proved during strain-induced PT. The degree of disorder is proposed to be used as a physical measure of plastic straining. This allows us to quantitatively separate the conventional plasticity and transformation-induced plasticity. Surprisingly, it is found that TRIP exceeds the conventional plasticity by a factor of 20. The cascade structural changes were revealed, defined as the reoccurrence of interacting processes including PTs, disordering, conventional plasticity, and TRIP. In comparison with hydrostatic loading, for the same degree of disorder, plastic shear indeed reduces the PT pressure (by a factor of 3-4) while causing a complete irreversible PT. The analytical results based on coupled strain-controlled kinetic equations for disorder and PT confirm our conclusions.

Aim: Celiac disease is characterized by life-long gluten intolerance. Clinical features of patients with celiac disease are variable. Studies about the prevalence of celiac disease in our country are scarce and there is no study on the prevalence of celiac disease in southern Iran. In the current study, clinical, laboratory and histo-logical features of 52 patients with celiac disease were evaluated., Methods: In a cross sectional study we retrospectively studied the characteristics of 52 celiac patients at Ahwaz JundiShapour University Hospitals (AJSUH) from November 1, 1999 to 1st Sep 2004. Intestinal biopsy and serum antigliadin and anti-endomysium antibodies were used for the diagnosis of patients. Mucosal lesions were classified according to the criteria of Marsh. Antigliadin antibodies were measured with a commercial enzyme-linked immunosorbent assay. Anti-endomysium antibodies were analyzed by indirect immunofluorescence with the use of a section of monkey esophagus. Routine hematological and biochemical analyses and measurement of immunoglobulin levels were undertaken., Results: Male: female ratio was 1.08. The mean +/- SD patient age was 21 +/- 4.5 years (range 10-70 years) and the most common symptoms were diarrhea and weight loss (78.8%) followed by fatigue (73.1%), pallor (65.4%), anorexia (40.4%), abdominal distention (32.7%), and failure to thrive (23.1%). Diarrhea and weight loss and fatigue were the most common findings. Iron deficiency anemia was found in 63.2% of patients and this became normal after adoption of a gluten-free diet in all patients. Immunoglobulin A, IgG antigliadin antibodies and IgA anti-endomysium antibodies were found in 33 and 48 cases, 78.8% and 85.4% of patients, respectively. Biopsy of the small intestine revealed that 90.4% of patients had typical lesions according to the Marsh classification., Conclusion: Although classical presentation was seen in most of the patients, atypical clinical manifestations of celiac disease should be kept in mind. In particular, patients with uncommon findings, such as short stature, and iron-deficiency anemia, should be screened for celiac disease. Further epidemiological studies in our area in the general population and in high risk groups seem to be indicated.

The effect of shear strain on the iron α-ε phase transformation has been studied using a rotational diamond anvil cell (RDAC). The initial transition is observed to take place at the reduced pressure of 10.8 GPa under pressure and shear operation. Complete phase transformation was observed at 15.4 GPa. The rotation of an anvil causes limited pressure elevation and makes the pressure distribution symmetric in the sample chamber before the phase transition. However, it causes a significant pressure increase at the centre of the sample and brings about a large pressure gradient during the phase transformation. The resistance to the phase interface motion is enhanced due to strain hardening during the pressure and shear operations on iron and this further increases the transition pressure. The work of macroscopic shear stress and the work of the pressure and shear stress at the defect tips account for the pressure reduction of the iron phase transition.

After immense amounts of research, the root cause for the significantly higher rates of anterior cruciate ligament (ACL) failure incidents in females as compared to males still remains unknown and the existing sex-based disparity has not diminished. To date, the possibility that the female ACL is mechanically weaker than the male ACL has not been directly investigated. Although it has been established in the literature that the female ACL is smaller in size, the differences in the structural and material properties of the ACL between sexes have not been studied. The aim of this cadaveric study was to determine if any sex-based differences in the tensile properties of the human ACL exist when considering age as well as ACL and body anthropometric measurements as covariates. Ten male and 10 female unpaired cadaveric knees (mean age 36.75 years) were used for this study. The geometry of the ACL (including length, cross-sectional area, and volume) was analyzed using a 3-D scanning system. The femur-ACL tibia complex was tested to failure along the longitudinal axis of the ligament in a tensile testing machine. The structural properties of the ACL as well as its mechanical properties were determined. Analysis of covariance was performed to assess the effect of sex on tensile properties. The female ACL was found to have a lower mechanical properties (8.3% lower strain at failure; 14.3% lower stress at failure, 9.43% lower strain energy density at failure, and most importantly, 22.49% lower modulus of elasticity) when considering age, ACL, and body anthropometric measurements as covariates.

Background: A significantly higher rate of anterior cruciate ligament injuries occurs in women involved in sports activities compared to the corresponding male population. Reasons for this disparity are not well understood; however, sex-based differences in the geometry of the anterior cruciate ligament, its morphologic characteristics, and the intercondylar notch size have been cited as possible factors., Hypotheses: (1) The anterior cruciate ligament in women has a shorter length, and smaller cross-sectional area and volume than that in men. (2) The female anterior cruciate ligament has a lower mass density. (3) Ligament size is proportional to notch width in both male and female populations., Study Design: Descriptive laboratory study., Methods: Using a 3-dimensional imaging system, the authors measured the geometric parameters, including length, area, and volume, of the anterior cruciate ligaments of 10 male and 10 female donors (all Caucasian). A digital image of the frontal plane of the knee flexed at 90 degrees was used to measure notch size. After the ligament's removal from its attachment sites, its mass was measured using a digital balance. Geometric parameters, mass density, and notch size were compared based on sex. Correlation analyses between ligament size and body anthropometric characteristics, and between notch size and ligament size were performed., Results: The anterior cruciate ligament in women was smaller in length, cross-sectional area, volume, and mass when compared to that in men. No significant difference in ligament mass density was found between the sexes. Also, no differences were found in notch geometry between male and female populations. A correlation between notch size and ligament size was found for men but not for women., Conclusions: Because the densities of female and male anterior cruciate ligaments appear to be similar, the smaller ligament size in women may contribute to their having a higher rate of ligament injuries. Anterior cruciate ligament size increases in proportion to notch width in men but not in women.

Background: A diverse range of values for the mechanical properties of the human patellar tendon has been reported in the literature. Donor age has been excluded as a factor that causes this disparity. However, the effects of donor sex and tissue mass density on the mechanical properties of the patellar tendon have not been established. More importantly, predictors of the mechanical properties of the patellar tendon, used as grafts in anterior cruciate ligament reconstruction surgeries, are not available., Methods: Uniaxial tension tests were performed to determine the effects of donor sex and mass density of the tissue on the mechanical properties of twenty unpaired human patellar tendons (10 male and 10 female; age, 17-50). Mass density of the patellar tendon was determined by measuring its volume and mass., Findings: No evidence of sex-based differences in any of the mechanical properties of the patellar tendon was found. The results revealed a diverse range of mass densities extending from a low of 0.76 g/cm(3) to a high of 2.68 g/cm(3) (250% difference). Furthermore, it was determined that the ultimate tensile strength (mean=58.71 MPa), elastic modulus (mean=507.38 MPa) and strain energy density (mean=4.59 MPa) of the patellar tendon were significantly correlated to its mass density. The tensile strength and elastic modulus were significantly higher for those patellar tendons having a mass density greater than 1.67 g/cm(3)., Interpretation: The mass density of the patellar tendon may be considered as an indicator of mechanical properties of the patellar tendon or graft quality.

Objective: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Its etiology and incidence differ according to geographic area. In the present study, we sought to identify risk factors for HCC among a group of patients with HCC in Southern Iran., Methods: During a 5-year period we retrospectively studied the characteristics of 71 HCC patients at Ahwaz Jundishapur University Hospital, Iran from February 1999 to August 2004. Blood samples and questionnaire data obtained from 71 (45 male and 26 female) incident cases of HCC, were pathologically diagnosed. Sera were tested for hepatitis B surface antigen and antibodies to hepatitis B core antigen, anti-hepatitis C virus and serum ferritin, iron and alfa fetoprotein (AFP), by enzyme immunoassays and cuper study., Results: In 46.5% (33/71) of patients there was a history of chronic liver disease, and in 30 (42%) patients liver cirrhosis was documented. Of the 71 patients, 37 (52.1%) had hepatitis B, 6 (8.5%) had hepatitis C, and 2 (2.8%) had a history of excess alcohol intake. Of the 2 patients with a history of heavy alcohol intake, one had concomitant chronic viral hepatitis infection, and alcohol alone was the etiology of HCC in only one case (1.40%). No etiologic cause was identified in 23 cases (32.4%), there were 2 cases of diabetes mellitus. The value of AFP of >20 ng/ml was found in 29 cases (41%), varying from 24 ng/ml to 364 ng/ml (average 74.6 ng/ml)., Conclusions: In Southern Iran, the predominant etiology of HCC was hepatitis B, hepatitis C, but alcohol and metabolic diseases were only found in rare cases. Cryptogenic cases may be found in one fifth of patients hence, the contribution of virus infection, may have been underestimated in this area, which is based on serological testing only.

Objectives: Somatic deletion of chromosome 11q13 is the most frequent genetic aberration in parathyroid adenoma. To gain further insight into the genetic etiology of parathyroid tumor development, we examined a comprehensive gene expression profile of parathyroid adenomas and normal parathyroid tissues. The results were then evaluated with respect to differences between adenomas and normal parathyroid tissue, and to the presence of loss of heterozygosity (LOH) in chromosomal region 11q13., Design and Methods: Sporadic parathyroid adenomas and normal parathyroids were hybridized against HG-U95Av2 oligonucleotide arrays (Affymetrix) containing a total of 12,625 probe sets. Quantitative real-time PCR (QRT-PCR) was performed in a larger series of parathyroid adenomas, in order to con-firm the microarray results., Results: Cyclin D1 and c-Jun showed increased expression in adenomas vs normal parathyroids by microarray analysis and QRT-PCR, suggesting an oncogenic role of these genes in parathyroid tumor development. At unsupervised hierarchical clustering, the adenomas fell into two groups: Group I adenomas were characterized by 11q13 LOH, while Group II adenomas lacked this abnormality. In addition, a t-test analysis identified largely non-overlapping genes with differential expression in the tumors subgroups; e.g. in Group I tumors the putative oncogene ENC 1 was found highly over-expressed vs Group II adenomas., Conclusions: The microarray analyses revealed partly distinctive and partly common expression profiles in parathyroid adenomas with and without 11q13 LOH. In addition, approximately half of the under-expressed genes were mapped to chromosome 11, in agreement with a dose effect following loss of this chromosome.

Background: Germline alterations in cyclin-dependent kinase inhibitor 2A (CDKN2A) are important genetic factors in familial predisposition to melanoma. Activating mutations of the NRAS proto-oncogene are among the most common somatic genetic alterations in cutaneous malignant melanomas. We investigated the occurrence of NRAS mutations in melanomas and dysplastic nevi in individuals with germline CDKN2A mutations., Methods: Genomic DNA was extracted from 39 biopsy samples (including primary melanomas, metastatic melanomas, and dysplastic nevi) from 25 patients in six Swedish families with a hereditary predisposition to melanoma who carried germline CDKN2A mutations. DNA was also extracted from 10 biopsy samples from patients with sporadic melanomas. NRAS was analyzed using polymerase chain reaction, single-strand conformation polymorphism analysis, and nucleotide sequence analysis. Differences in NRAS mutation frequency between hereditary and sporadic melanomas were analyzed by the chi-square test. All statistical tests were two-sided., Results: Activating mutations in NRAS codon 61, all of which were either CAA(Gln)-AAA(Lys) or CAA(Gln)-CGA(Arg) mutations, were found in 95% (20/21) of primary hereditary melanomas but in only 10% (1/10) of sporadic melanomas (P<.001). Multiple activating NRAS mutations were detected in tumor cells from different regions of individual primary melanomas in nine patients. Activating mutations that were detected in the primary melanomas of these patients were also retained in their metastases. NRAS mutations at sites other than codon 61 were also present in the primary melanomas, indicating genetic instability of this locus. NRAS codon 61 mutations were also detected in dysplastic nevi and in an in situ melanoma, suggesting a role for such mutations during early melanoma development., Conclusions: The high frequency of NRAS codon 61 mutations detected in these hereditary melanomas may be the result of a hypermutability phenotype associated with a hereditary predisposition for melanoma development in patients with germline CDKN2A mutations.

The CDKN2A locus on human chromosome 9p21 encodes two proteins, p16 and p14ARF, that mainly regulate cell cycle progression and cell survival via the pRb and p53 pathways, respectively. Germline mutations in CDKN2A have been linked to development of cutaneous melanoma in some families with hereditary melanoma. Due to overlapping open reading frames in exon 2, some mutations in this exon affect both p16 and p14ARF. We previously reported a 24bp deletion in CDKN2A exon 2 in a patient with multiple primary melanomas and melanoma heredity. To further clarify the possible role of the 24bp deletion for melanoma development, especially with respect to p14ARF, we have studied the cellular distribution and function of the resulting p14ARF del (77-84) and p16 del (62-69) mutant proteins. We found that p14ARF del (77-84) had decreased nucleolar localization, and was less efficient than wt p14ARF in stabilizing p53, inducing G1 cell cycle arrest, and inhibiting colony formation. The p16 del (62-69) mutant localized predominantly to the cytoplasm, did not induce G1 cell cycle arrest, and failed to suppress colony formation. We conclude that p14ARF del (77-84) has retained the ability to stabilize MDM2 and p53, but that it is less potent than wt p14ARF. This partial functional defect may complement the clearly defective p16 del (62-69) mutant and thus contribute to melanoma development in patients carrying the 24bp deletion in CDKN2A.

Germline mutations in the CDKN2A tumor suppressor gene located on 9p21 have been linked to development of melanomas in some families. A germline 3-bp insertion in exon 2 of CDKN2A, leading to an extra arginine at codon 113 (113insR), has been identified in 17 Swedish melanoma families. Analysis of 10 microsatellite markers, spanning approximately 1 Mbp in the 9p21 region, showed that all families share a common allele for at least one of the markers closest to the CDKN2A gene, suggesting that the 113insR mutation is an ancestral founder mutation. Differences in the segregating haplotypes, due to meiotic recombinations and/or mutations in the short-tandem-repeat markers, were analyzed further to estimate the age of the mutation. Statistical analysis using a maximum likelihood approach indicated that the mutation arose 98 generations (90% confidence interval: 52-167 generations), or approximately 2,000 years, ago. Thus, 113insR would be expected to have a more widespread geographic distribution in European and North American regions with ancestral connections to Sweden. Alternatively, CDKN2A may lie in a recombination hot spot region, as suggested by the many meiotic recombinations in this narrow approximately 1-cM region on 9p21., (Copyright 2001 Wiley-Liss, Inc.)

Germ-line CDKN2A mutations are present in some kindreds with hereditary cutaneous melanoma, and in Sweden a founder mutation with an extra arginine in codon 113 (113insR) has been identified. We screened 80 individuals with at least two primary cutaneous melanomas, who were identified mainly by a search of a regional cancer registry, for germ-line CDKN2A mutations. In nine patients, CDKN2A alterations that may contribute to melanoma predisposition were detected. In six individuals with a family history of melanoma, the 113insR founder mutation was present. One patient, who also had a family history of melanoma, had a 24-bp deletion that included codons 62-69. An in vitro binding assay established that the resulting mutant p16 protein was unable to bind cyclin-dependent kinase 4 and cyclin-dependent kinase 6. Two patients without a family history of melanoma had CDKN2A alterations: (a) one had a mutation in the 5' noncoding sequence (-14C/T); and (b) the other had an insertion of an extra T in codon 28, which results in a stop signal in codon 43. The median age at diagnosis of the first melanoma was significantly lower, the number of primary melanomas was significantly higher, and the presence of a family history of melanoma was significantly more common in patients with CDKN2A mutations than in those without germ-line mutations. The proportion of CDKN2A mutation carriers was significantly higher among patients treated for three or more primary melanomas compared with those with two tumors only. We conclude that mutation screening of individuals with multiple primary melanomas is a useful strategy to identify new melanoma kindreds with CDKN2A germ-line mutations.

The CDKN2A gene encodes the cell cycle inhibitor p16/ INK4A, which is involved in familial cutaneous melanoma. We have studied five Swedish familial melanoma kindreds characterized by germline mutations in CDKN2A and dysplastic naevus syndrome (DNS). We found significant correlations between germline CDKN2A mutations and melanoma and between DNS phenotype and melanoma, respectively. There was also a correlation between mutation status and the presence of DNS. In CDKN2A mutation carriers, all cases of early-onset melanoma occurred in DNS individuals, and the mean age at melanoma diagnosis was significantly lower in individuals with DNS than in those without a confirmed DNS phenotype. In one family where the proband had a P48L mutation in CDKN2A exon 1, the DNS phenotype was studied in detail. In vitro binding experiments established that the P48L mutant protein does not bind to cdk4 or cdk6 and thus is functionally abnormal. Furthermore, we demonstrated loss of heterozygosity at markers on chromosome 9p flanking the CDKN2A locus in a primary melanoma and a metastasis from the proband. Our results are consistent with the hypothesis that germline CDKN2A mutations and DNS both contribute to the predisposition to melanoma and may lead to the development of early-onset melanoma when present in the same individual.

Rat embryo fibroblasts (REFs) are inefficiently transformed by RAS-oncogenes. Induction of p16INK4A expression by RAS has been suggested to contribute to this resistance. Glucocorticoid hormones, (DEX), enhance REF transformation by RAS and facilitates the isolation of transformed and immortal cell lines. We show that DEX induced cell proliferation is paralleled by a decrease in Cdkn2a gene transcripts, suggesting a mechanism for hormone promotion. The mechanisms of progression into hormone independent cell lines were examined. Twenty-two of 30 clones which reached a population size of approximately 10(6) cells could be established as cell lines. All lines studied showed homozygous deletions of the Cdkn2 loci (Cdkn2a and Cdkn2b) on RNO5. LOH was found for all RNO5 genetic markers examined in 7 of 19 cell lines, suggesting non-disjunction events. In the remaining 12 cell lines, both copies of Cdkn2 appeared to be lost by deletions/rearrangements, some of which could by demonstrated by karyotype analysis. We conclude that (i) clonal expansion of RAS-transfected REF by DEX is paralleled by down-regulation of Cdkn2a expression; (ii) homozygous deletion of Cdkn2 were estimated to occur at a frequency of 2 x 10(-8)/cell/generation or higher, and (iii) deletion/rearrangements and nondisjunction appear to be the main mechanisms leading to deletion of Cdkn2.