Application of discriminant analysis and quantitative cytologic examination to gastric lesions

OBJECTIVE: To investigate of the potential value of morphometry and discriminant analysis for the classification of benign and malignant gastric cells and lesions. STUDY DESIGN: The data set consisted of 13,300 cells from 120 cases composed of 30 cases of cancer, 26 cases of gastritis and 64 cases of ulcer according to the final histologic diagnosis. The cytologic diagnosis was divided into 5 categories (gastritis, ulcer, inflammatory dysplasia, cancer and true dysplasia). Classification was attempted at 2 levels: the cell level to classify individual cells and the case level to classify individual cases. For the cellular classification the measured cells from 50% of available cases were selected as a training set to construct a model. The cells from the remaining cases were used as a test set to validate the model. Similarly for case classification, the same 50% of cases that were used for cell classification were used as a training set and the remaining cases as a test set. Images of routinely processed gastric smears stained by the Papanicolaou technique were analyzed by a customized image analysis system. RESULTS: Application of discriminant analysis on the test set gave correct classification of 98.4% of benign cells and 67.1% of malignant cells. On case classification, 100% accuracy was achieved for benign and malignant cases, both for the training and test sets. CONCLUSION: The application of discriminant analysis described in this paper could produce significant classification results at the cellular and individual case level.     

Description of the first two seemingly unrelated Greek Cypriot families with a common C618R RET proto-oncogene mutation

Germ-line mutations of the RET proto-oncogene cause three different cancer syndromes: multiple endocrine neoplasia type 2A (MEN2A), multiple endocrine neoplasia type 2B, and familial medullary thyroid carcinoma (FMTC). The objective of the present study was the clinical and molecular characterization of the first two Greek Cypriot families diagnosed with MEN2A and FMTC. The clinical diagnosis of the probands was based on clinical presentation and supported with laboratory findings (calcitonin and carcinoembryonic antigen tumor marker levels). We screened the RET gene by direct DNA sequencing of exons 10, 11, and 16 using genomic DNA as templates. After identification of the mutation, we also developed the amplification refractory mutation system (ARMS) as an alternative method to direct sequencing for genetic diagnosis of 22 additional individuals from both families. We identified the germ-line missense mutation T --> C of codon 618 of exon 10 (C618R) in the probands of both families. By using ARMS, two members of the MEN2A family and five members of the FMTC family were also found positive for the C618R mutation. These are the first seemingly unrelated families in Cyprus investigated clinically and molecularly in detail and shown to transmit this common RET proto-oncogene mutation.     

Mice with targeted mutation of peroxiredoxin 6 develop normally but are susceptible to oxidative stress

Reactive oxygen species, especially hydrogen peroxide, are important in cellular signal transduction. However, excessive amounts of these species damage tissues and cells by oxidizing virtually all important biomolecules. Peroxiredoxin 6 (PRDX6) (also called antioxidant protein 2, or AOP2) is a novel peroxiredoxin family member whose function in vivo is unknown. Through immunohistochemistry, we have determined that the PRDX6 protein was widely expressed in every tissue examined, most abundantly in epithelial cells. It was found in cytosol, but not in membranes, organelles, and nuclei fractions. Prdx6 mRNA was also expressed in every tissue examined. The widespread expression of Prdx6 suggested that its functions were quite important. To determine these functions, we generated Prdx6-targeted mutant (Prdx6-/-) mice, confirmed the gene disruption by Southern blots, PCR, RT-PCR, Western blots, and immunohistochemistry, and compared the effects of paraquat, hydrogen peroxide, and t-butyl hydroperoxide on Prdx6-/- and wild-type (Prdx6+/+) macrophages, and of paraquat on Prdx6-/- and Prdx6+/+ mice. Prdx6-/- macrophages had higher hydrogen peroxide levels, and lower survival rates; Prdx6-/- mice had significantly lower survival rates, more severe tissue damage, and higher protein oxidation levels. Additionally, there were no differences in the mRNA expression levels of other peroxiredoxins, glutathione peroxidases, catalase, superoxide dismutases, thioredoxins, and glutaredoxins between normal Prdx6-/- and Prdx6+/+ mice and those injected with paraquat. Our study provides in vivo evidence that PRDX6 is a unique non-redundant antioxidant that functions independently of other peroxiredoxins and antioxidant proteins.     

Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Sporotrichum thermophile

An endo-beta-1,4-xylanase (1,4-beta-D-xylan xylanoxydrolase, EC 3.2.1.8) present in culture filtrates of Sporotrichum thermophile ATCC 34628 was purified to homogeneity by Q-Sepharose and Sephacryl S-200 column chromatographies. The enzyme has a molecular mass of 25,000 Da, an isoelectric point of 6.7, and is optimally active at pH 5 and at 70 degrees C. Thin-layer chromatography (TLC) analysis showed that endo-xylanase liberates mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methylglucuronoxylan and rhodymenan (a beta-(1-->4)-beta(1-->3)-xylan). Also, the enzyme releases an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. Analysis of reaction mixtures by high performance liquid chromatography (HPLC) revealed that the enzyme cleaves preferentially the internal glycosidic bonds of xylooligosaccharides, [1-3H]-xylooligosaccharides and xylan. The enzyme also hydrolyses the 4-methylumbelliferyl glycosides of beta-xylobiose and beta-xylotriose at the second glycosidic bond adjacent to the aglycon. The endoxylanase is not active on pNPX and pNPC. The enzyme mediates a decrease in the viscosity of xylan associated with a release of only small amounts of reducing sugar. The enzyme is irreversibly inhibited by series of omega-epoxyalkyl glycosides of D-xylopyranose. The results suggest that the endoxylanase from S. thermophile has catalytic properties similar to the enzymes belonging to family 11.     

Spectroimmunochemistry using colloidal gold bioconjugates

Using surface-enhanced infrared absorption (SEIRA) spectroscopy of dry films of colloidal gold (CG) bioconjugates with protein A, it is shown that certain characteristic bands of the protein (e.g., amide I, amide II and some other vibration modes) are essentially affected by the metal surface. Thus, the method may be used for controlling the quality of such bioconjugates. Moreover, it is demonstrated that the biospecific reaction of protein A attached to CG particles with human immunoglobulin G (IgG) results in further essential changes in SEIRA spectra, providing a means for an easy and rapid IR spectroscopic detection of biospecific immunochemical interactions (i.e., spectroimmunochemistry). The results obtained can form a basis for developing test systems for detecting various biospecific interactions.     

Cloning, chromosomal organization and expression analysis of Neurl, the mouse homolog of Drosophila melanogaster neuralized gene

The Drosophila neuralized (neur) gene belongs to the neurogenic group of genes involved in regulating cell-cell interactions required for neural precursor development. neur mutant phenotypes include strong overcommitment to neural fates at the expense of epidermal fates. The human neuralized homolog (NEURL) has been recently determined and found to map to chromosome 10q25.1 within the region frequently deleted in malignant astrocytomas. Because of its potential importance in developmental processes, we analyzed the structure of the mouse homolog, Neurl, and its expression pattern in embryonic tissues. Neurl activity is detected from early developmental stages in several tissues and organs including neural tissues, limbs, the skeletal system, sense organs and internal organs undergoing epithelial-mesenchymal interactions. Neurl encodes a polypeptide associated with the plasma membrane but also detected in the cytoplasm. Similarly to the Drosophila gene, mammalian neuralized may code for an important regulatory factor.     

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation

The Bcl-2 homology 3 (BH3) domain of prodeath Bcl-2 family members mediates their interaction with prosurvival Bcl-2 family members and promotes apoptosis. We report that survival factors trigger the phosphorylation of the proapoptotic Bcl-2 family member BAD at a site (Ser-155) within the BAD BH3 domain. When BAD is bound to prosurvival Bcl-2 family members, BAD Ser-155 phosphorylation requires the prior phosphorylation of Ser-136, which recruits 14-3-3 proteins that then function to increase the accessibility of Ser-155 to survival-promoting kinases. Ser-155 phosphorylation disrupts the binding of BAD to prosurvival Bcl-2 proteins and thereby promotes cell survival. These findings define a mechanism by which survival signals inactivate a proapoptotic Bcl-2 family member, and suggest a role for 14-3-3 proteins as cofactors that regulate sequential protein phosphorylation events.