H-ras oncogene mutation in dedifferentiated liposarcoma. Polymerase chain reaction-restriction fragment length polymorphism analysis.
Point mutations of the ras gene family (K-ras, H-ras, and N-ras) are thought to be involved in the development of a variety of human tumors. Dedifferentiated liposarcoma is characterized by the coexistence of well-differentiated (WD) and high-grade anaplastic (HG) components. The presence of point mutations at codons 12 and 13 of the H-ras gene was studied in 34 liposarcomas, comprising 15 well-differentiated liposarcomas and 19 dedifferentiated liposarcomas, and in 8 storiform-pleomorphic type malignant fibrous histiocytomas (MFHs) using polymerase chain reaction-restriction fragment length polymorphism and direct sequencing analysis. The 2 components of dedifferentiated liposarcoma were analyzed independently. H-ras mutations were seen only in dedifferentiated liposarcomas (4/19 [21%]), 1 in WD components and 3 in HG components. The mutation was not seen in any of 15 cases of well-differentiated liposarcoma. MFHs showed an H-ras mutation in 1 (12%) of 8 cases. Our results seem to suggest that the H-ras mutation is a relatively uncommon event in dedifferentiated liposarcoma, which may demonstrate an epiphenomenon of dedifferentiation in dedifferentiated liposarcoma. (+info)
Prior exposure to aged and diluted sidestream cigarette smoke impairs bronchiolar injury and repair.
The bronchiolar injury/repair response to naphthalene (NA) in mice includes acute distal airway epithelial injury that is followed by epithelial proliferation and redifferentiation, which result in repair of the epithelium within 14 days. To test whether prior exposure to aged and diluted sidestream cigarette smoke (TS) would alter the injury/repair response of the airway epithelium, adult mice were exposed to either filtered air (FA) or smoke for 5 days before injection with either corn oil carrier (CO) or naphthalene. Mice were killed 1 and 14 days after naphthalene injury. Lung and lobar bronchus were examined and measured using high-resolution epoxyresin sections. The control group (FACOFA) that was exposed to filtered air/corn oil/filtered air contained airway epithelium similar to untreated controls at all airway levels. The group exposed to tobacco smoke/corn oil/filtered air (TSCOFA) contained some rounded cells in the small airways and some expansion of the lateral intercellular space in the larger airways. Necrotic or vacuolated cells were not observed. As expected, the epithelium in the group exposed to filtered air/naphthalene/filtered air (FANAFA) contained many light-staining vacuolated Clara cells and squamated ciliated cells within distal bronchioles during the acute injury phase. Repair (including redifferentiation of epithelial cells and restoration of epithelial thickness) was nearly complete 14 days after injury. The extent of Clara cell injury, as assessed in lobar bronchi, was not different between the four groups. Although the FANAFA group contained greater initial injury in the distal airways at 1 day, the group exposed to tobacco smoke/naphthalene/filtered air (TSNAFA) had the least amount of epithelial repair at 14 days after naphthalene treatment; many terminal bronchioles contained abundant squamated undifferentiated epithelium. We conclude that tobacco smoke exposure prior to injury (1) does not change the target site or target cell type of naphthalene injury, since Clara cells in terminal bronchioles are still selectively injured; (2) results in slightly diminished acute injury from naphthalene in distal bronchioles; and (3) delays bronchiolar epithelial repair. (+info)
Analysis of intratumoral heterogeneity of chromosome 3p deletions and genetic evidence of polyclonal origin of cervical squamous carcinoma.
Investigation on intratumoral genetic heterogeneity provides an important insight into the roles of genetic alterations in human carcinogenesis and clues to clonal origin of tumors. Intratumoral heterogeneity of genetic changes of cervical cancer has not been described so far. In this study, we analyzed the intratumoral heterogeneity of chromosome 3p deletions and X-chromosome inactivation patterns in multiple microdissected samples from each individual cervical cancer, attempting to understand the roles of 3p deletions in development of cervical cancer and its clonal origin. Totally, 120 normal and lesional samples from 14 cases of fresh cervicalcancers were analyzed. Frequency and patterns of allelic losses of 3p were assessed by polymerase chain reaction (PCR) amplification of 12 microsatellite markers flanking the frequently deleted regions of 3p, followed by Genescan analysis in an ABI 377 DNA sequencer. Loss of heterozygosity was recorded as heterogeneous pattern (LOH present in parts of samples or LOH involving different alleles among different samples) and homogeneous pattern (LOH involving identical alleles in all samples from the tumor). Allelic loss affecting at least one marker was detected in 8 of 14 cases (57%). Allelic losses, both homogeneous and heterogeneous, were frequently detected at FHIT gene region (D3S1300, 40% and 60%; D3S4103, 27.3% and 54.6%), 3p21.3-21.2 (D3S1478, 27.3% and 45.5%), and 3p24.2-22 (D3S1283, 30% and 50%). Seven of eight LOH-positive tumors exhibited homogeneous allelic loss involving at least one of these three 3p loci. Allelic losses were present in the CIN lesions synchronous with invasive lesions positive for LOH. Our findings suggest essential roles of genes on these 3p loci, particularly the FHIT gene in participating in clonal selection and early development of cervical cancer. Most interestingly, with the combination of LOH analysis and X-chromosome inactivation analysis, we provided the first clear genetic evidence of polyclonal origin of cervical invasive cancer in two of eight cases. This finding strongly suggests the importance of field defect (possible human papilloma virus) in cervical carcinogenesis. (+info)
Male infertility and intracytoplasmic sperm injection (ICSI).
Micro-assisted fertilization in the form of intracytoplasmic sperm injection (ICSI) has truly revolutionised the treatment options for couples with impaired semen quality, and those with both obstructive and non-obstructive azoospermia. In general, the major issues which relate to the success of ICSI are those related to the success of conventional IVF, and the high multiple pregnancy rate remains a major cause for concern. There is growing evidence that the short-term health of ICSI offspring is relatively unremarkable, but our growing understanding of the genetic basis of much of male subfertility and of the impaired genomic integrity which characterises the oligozoospermic phenotype indicate a cautious approach to the longer term health of ICSI offspring. (+info)
Follicular lymphoma with marginal zone differentiation: microdissection demonstrates the t(14;18) in both the follicular and marginal zone components.
On occasion, follicle center lymphomas (FCL) may contain a marginal-zone (MZ) component in which the interfollicular lymphoid cells take on an MZ cell morphology. In the past, these have been termed composite lymphomas. However, recent studies suggest that the two components are clonally related. It is unknown whether the bcl-2 translocation present in most FCLs is present in the cells that demonstrate MZ cell morphology. We have identified three cases of low-grade FCL with a MZ component suitable for laser capture microdissection (LCM) of the two components. Cases were immunophenotyped in paraffin section with antibodies to CD10, CD20, bcl-2, and bcl-6. LCM was done to isolate cells from each component. Polymerase chain reaction for t(14;18) using primers to the major breakpoint region was performed on DNA extracts. The sensitivity of the PCR assay was decreased to 5%--10% follicle center cells in a background of reactive tonsil cells. All three cases showed different phenotypes in each component. The FCL component was positive for all four of the above markers, whereas the MZ component expressed only CD20 and bcl-2. Both components showed t(14;18) amplicons of identical size, with the MZ component signal being stronger than the 5%--10% sensitivity control, suggesting that the signal was not from rare, contaminating FCL cells. These results confirm that both components are clonally related and support the theory that these are indeed FCLs with MZ differentiation (that retain the t(14;18)) rather than the reverse, MZ lymphoma with follicle center differentiation. (+info)
Controlled rotation of optically trapped microscopic particles.
We demonstrate controlled rotation of optically trapped objects in a spiral interference pattern. This pattern is generated by interfering an annular shaped laser beam with a reference beam. Objects are trapped in the spiral arms of the pattern. Changing the optical path length causes this pattern, and thus the trapped objects, to rotate. Structures of silica microspheres, microscopic glass rods, and chromosomes are set into rotation at rates in excess of 5 hertz. This technique does not depend on intrinsic properties of the trapped particle and thus offers important applications in optical and biological micromachines. (+info)
Mutational analysis of the 5' noncoding region of the bcl-6 gene in primary gastric lymphomas.
Bcl-6 mRNA and protein are frequently expressed in the transformed counterparts of the germinal center B-cells, diffuse large B-cell lymphoma and follicular lymphoma, irrespective of the gene rearrangements. Most of the primary gastric lymphomas are thought to be of mucosa-associated lymphoid tissue (MALT) origin, and neither bcl-6 gene rearrangement nor protein expression is found in low-grade gastric lymphomas of the MALT type as in normal marginal zone cells. However, bcl-6 protein expression was identified in high-grade gastric lymphomas, suggesting its role in high-grade transformation. In this study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis for bcl-6 primer was performed in order to ascertain the molecular mechanisms of bcl-6 protein expression in primary gastric lymphomas. A total 31 cases of gastric lymphoma were classified into low-grade gastric lymphomas of MALT type (n = 13), high-grade gastric lymphomas of MALT type (n = 6) and gastric diffuse large B-cell lymphomas (n = 12). Bcl-6 mutations were observed in 11 of 13 (84.6%) low-grade gastric lymphomas of the MALT type and in 8 of 12 (66.7%) diffuse large B-cell gastric lymphomas. In 6 cases of the high-grade gastric lymphomas of the MALT type, both the low- and high-grade components demonstrated the same frequency (3/6, 50%) of mutations. The tissue obtained from the marginal zone of Peyer's patch by microdissection technique revealed no bcl-6 mutations by the PCR-SSCP analysis. These findings suggest that the acquisition process of bcl-6 mutations by the marginal zone cells may be involved in the lymphomagenesis of the stomach, but our data does not explain the reason why bcl-6 protein is expressed only in high-grade gastric lymphomas. (+info)
Single-cell dissection and microdroplet chemistry.
The unique roles of individual cells may be critical to the physiology of an organism. In such cases, micromethods are essential to elucidating the molecular biology, biochemistry and biophysics of the specialized cells or even subcellular compartments of the important cells. The great proliferation of micromethods testifies to their value and no single review can be comprehensive. This review therefore provides only a generalized overview of one approach, namely dissection that provides a pure sample for subsequent extraction and analysis by microdroplet chemistry. As a means of illustrating the utility of this approach, an application-study of the interaction of cytosolic malate concentration and guard-cell phosphoenolpyruvate carboxylase-is provided. (+info)