NF1 gene analysis based on DHPLC

The high mutation rate at the NF1 locus results in a wide range of molecular abnormalities. The majority of these mutations are private and rare, generating elevated allelic diversity with a restricted number of recurrent mutations. In this study, we have assessed the efficacy of denaturing high-performance liquid chromatography (DHPLC), for detecting mutation in the NF1 gene. DHPLC is a fast and highly sensitive technique based on the detection of heteroduplexes in PCR products by ion pair reverse-phase HPLC under partially denaturing conditions. We established theoretical conditions for DHPLC analysis of all coding exons and splice junctions of the NF1 gene using the WAVEmaker software version 4.1.40 and screened for mutations a panel of 40 unrelated NF1 patients (25 sporadic and 15 familial), genetically uncharacterized. Disruptive mutations were identified in 29 individuals with an overall mutation detection rate of 72.5%. The mutations included eight deletions (exons 4b, 7, 10a, 14, 26, and 31), one insertion (exon 8), nine nonsense mutation (exons 10a, 13, 23.1, 27a, 29, 31, and 36), six missense mutations (exons 15, 16, 17, 24, and 31), four splice errors (exons 11, 14, 36, and 40) and a complex rearrangement within exon 16. Eighteen (62%) of the identified disruptive mutations are novel. Seven unclassified and three previously reported polymorphisms were also detected. None of the missense mutations identified in this study were found after screening of 150 controls. Our results suggest that DHPLC provides an accurate method for the rapid identification of NF1 mutations.     

CDK9/CYCLIN T1 expression during normal lymphoid differentiation and malignant transformation

CDK9 is a member of the CDC2-like family of kinases. Its cyclin partners are members of the CYCLIN T family (T1, T2a, and T2b) and CYCLIN K. The CDK9/CYCLIN T1 complex is very important in the differentiation programme of several cell types, controlling specific differentiation pathways. Limited data are available regarding the expression of CDK9/CYCLIN T1 in haematopoietic and lymphoid tissues. The aim of this study was to analyse the expression of the CDK9/CYCLIN T1 complex in lymphoid tissue, in order to assess its role in B- and T-cell differentiation and lymphomagenesis. CDK9/CYCLIN T1 expression was found by immunohistochemistry in precursor B and T cells. In peripheral lymphoid tissues, germinal centre cells and scattered B- and T-cell blasts in interfollicular areas expressed CDK9/CYCLIN T1, while mantle cells, plasma cells, and small resting T-lymphocytes displayed no expression of either molecule. CDK9/CYCLIN T1 expression therefore appears to be related to particular stages of lymphoid differentiation/activation. CDK9 and CYCLIN T1 were highly expressed in lymphomas derived from precursor B and T cells, from germinal centre cells, such as follicular lymphomas, and from activated T cells (ie anaplastic large cell lymphomas). Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma also showed strong nuclear staining. Diffuse large B-cell, Burkitt's lymphomas, and peripheral T-cell lymphomas, among T-cell lymphoproliferative disorders, showed a wide range of values. No expression of CDK9 or CYCLIN T1 was detected in mantle cell and marginal zone lymphomas. However, at the mRNA level, an imbalance in the CDK9/CYCLIN T1 ratio was found in follicular lymphoma and diffuse large B-cell lymphomas with germinal centre phenotype, and in the cell lines of classical Hodgkin's lymphomas, Burkitt's lymphomas, and anaplastic large cell lymphoma, in comparison with reactive lymph nodes. These results suggest that the CDK9/CYCLIN T1 complex may affect the activation and differentiation programme of lymphoid cells. The molecular mechanism through which the CDK9/CYCLIN T1 complex is altered in malignant transformation needs to be elucidated.     

Spontaneous and MNNG‐induced reversion of an EGFP construct in HeLa cells: An assay for observing mutations in living cells by fluorescent microscopy

A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2‐654 intron, was studied without treatment and after treatment with a single standard dose of 15 μM of N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). This assay was done in order to prove that such a construct can revert by a variety of mechanisms and that it produces a visible phenotype, i.e., green fluorescence. The system permits visual detection of living mutant cells among a background of non‐mutant cells and does not require a selective medium. The results show that the construct reverts by large deletions (–62, –100, and –162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at purines (G₆₅₂, G₆₅₃, A₆₅₅, G₅₇₉), and a pyrimidine (T₆₅₄) between nucleotide positions 579 and 837. Splice‐site mutations were recovered, and some of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wide variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay. Hum Mutat 18:526–534, 2001. © 2001 Wiley‐Liss, Inc.     

Hepatic lymphoid aggregates in chronic hepatitis C and mixed cryoglobulinemia

Summary We have examined the clinical (virological and immunological), histological and immunohistochemical features of liver lymphoid nodules in hepatitis C virus-positive (HCV⁺)/mixed cryoglobulinemia (type II and III) and chronic hepatitis C. The clinical features of liver disease were found to be similar in all patients. In all these groups, liver lymphoid nodules were observed to a similar extent, being more frequent in earlier phases of liver disease and less in more advanced stages. These data were confirmed by studies in serial biopsy samples taken from individual patients with type II mixed cryoglobulinemia; the loss of lymphoid nodules with progression to more advanced histological stages of disease in these patients was accompanied by a decrease of the serum levels of cryoglobulins (although not statistically significant). By immunohistochemical analysis, the liver lymphoid nodules contained predominantly B cells with a CD5⁺/bcl2⁺/Ki67^– phenotype, which were always polyclonal in type III mixed cryoglobulinemia and chronic hepatitis C, and monoclonal in type II mixed cryoglobulinemia. These immunological features were consistent with an active role of the immune system in HCV-associated liver necro-inflammation. Only in type II mixed cryoglobulinemia was there a clonal restriction of B cells. The immunological profile (autoantibodies) and viral genotypes were examined in some patients, but no significant correlation with clinical and immunohistochemical findings was found; however, the prevalence of genotype 2a was significantly higher in type II mixed cryoglobulinemia than in type III and chronic hepatitis without cryoglobulinemia.     

The nature of high frequency sister chromatid exchange cells (HFCs)

We employed the three-way differential staining technique (TWD),which allows SCEs to be distinguished on a per generation basisby scoring third metaphases (M3), in order to study the spontaneouslevels of SCEs in normal and high frequency cells (HFCs) thatoccurred in the first (S1), second (S2) and third (S3) S phases.Fifty one of 900 lymphocytes from 37 healthy donors were definedas HFCs by calculating the 95th percentile of the distributionof SCEs in S1 + S2. ‘Normal’ cells presented almostthe same number of SCEs after the first, second and third cellcycles (SCE averages of 2.43, 2.04 and 3.53 respectively). Incontrast, HFCs showed a higher SCE count in SI, which decreasedrapidly through the cycles and reached baseline level at S3(SCE averages of 7.18, 4.29 and 3.45 respectively). This wouldsuggest that the lesions responsible for the higher SCE frequencyin HFCs were effectively removed after two cell cycles and stronglysupport the hypothesis that HFCs are lymphocytes which accumulatehigher levels of DNA lesions through time. ¹To whom correspondence should be addressed     

Vascular pulsations stimulating nitric oxide release during cyclic exercise may benefit health: a molecular approach (review)

It is widely assumed that all exercise, regardless of the degree of difficulty or strenuousness, is good (no pain-no gain). In this speculative review of the literature and our research findings we highlight the fact that strenuous exercise taken to the extreme initiates an immune and vascular proinflammatory situation. However, mild cyclic exercise appears to produce health benefits for an individual. In part, this is due to vascular cyclic pulsations, occurring in mild exercise, stimulating constitutive nitric oxide synthase derived nitric oxide release. This in turn down-regulates vascular endothelial cells and immunocytes, as well as their interaction and inhibits the disassociation of NF-kappaB, preventing the production of proinflammatory cytokines. The nitric oxide so generated may even scavenge excess free radicals, preventing tissue damage. Prolonged strenuous exercise appears to limit these positive phenomena because of the maintained and prolonged high blood pressure that reduces the cyclic pulsations, limiting nitric oxide production. We further note that pathological conditions, i.e., Parkinson's disorder, may benefit from mild exercise, i.e., cyclic nitric oxide production, since the inactivity associated with this disease may lead to compromised nitric oxide production, initiating a progressive deterioration of tissues, including peripheral adrenergic neurons, due to a lack of adequate basal nitric oxide levels required to maintain the vascular microenvironment in a mild state of inhibition. We conclude that mild exercise represents an alternate and economical therapy to preserve health and/or diminish the rate of decline of the normal physiological processes that may even be associated with aging.     

Association of single nucleotide polymorphisms of the interleukin-10 promoter gene and susceptibility to primary biliary cirrhosis: immunogenetic differences in Italian and Japanese patients

Several lines of data suggest that genetic factors play an important role in the onset and/or progression of primary biliary cirrhosis (PBC). Since PBC is an autoimmune disease, it is reasoned to assume that genes encoding cytokines may confer susceptibility to disease. Amongst these factors, interleukin-10 (IL-10) has received significant attention. The promoter region of IL-10 gene has three single nucleotide polymorphisms (SNPs) at positions -1082, -819 and -592. To elucidate the association of the three SNPs of IL-10 promoter region with susceptibility of PBC in two different genetic populations, 159 unrelated patients with PBC (94 Italian and 65 Japanese) and 143 local controls (72 Italian and 71 Japanese) were enrolled. SNPs were determined using allele-specific PCR/RFLP. In Italian PBC patients, the frequency of homozygosity for G/G at position -1082 was significantly higher than that of local controls (p < 0.041, OR = 2.44, 95% C.I.; 1.02-5.86). The frequencies of haplotype GCC in PBC patients, possibly linked to higher IL-10 production, were also significant higher than local controls (p < 0.033). However, in Japanese population, there were no significant differences in the three SNPs and haplotypes between PBC patients and controls. Excessive production of IL-10 may play an important role in some populations in modulating the onset of PBC. Further, immunogenetic studies of PBC should take into account ethnic and geographic variations; this makes such studies in heterogeneous population, like the USA, more difficult.