Hemochromatosis (HFE) gene sequence analysis of formalin-fixed, paraffin-embedded liver biopsy specimens.

BACKGROUND: Hereditary hemochromatosis (HH) is a common disease predominantly characterized by mutations of the HFE gene. METHODS AND RESULTS: We investigated the utility of HFE gene sequence analysis in the diagnosis of HH in 61 prospectively accrued formalin-fixed, paraffin-embedded liver biopsy specimens with clinical or histologic features suggestive of HH. Mutations in codons 63 or 282 of the HFE gene were identified by direct sequencing; in 21 of these samples, quantitative hepatic iron testing was also performed. Changes characteristic of HH were present in 16 (26%) of the cases, and 54% of the cases showed HFE gene mutations. The most common alteration was homozygous mutation of codon 282 (11 cases, 18%), followed by the combined 63 + 282 heterozygous mutation (3 cases, 5%). Two cases (3%) showed biallelic mutation of codon 63. The other 28 cases (46%) showed no sequence abnormalities. Weak iron staining did not exclude HH; intense staining did not reliably predict HH. CONCLUSION: When HH is clinically and/or histologically suspected, HFE gene sequencing of formalin-fixed, paraffin-embedded liver biopsy specimens is a rapid and cost-effective approach to genotypic diagnosis of HH.

Expression of an isoform of the novel signal transduction protein ST5 is linked to cell morphology.

The human ST5 gene is expressed as 4.6, 3.1 and 2.8 kb transcripts encoding putative 126, 82 and 70 kDa proteins that function in the MAP kinase signaling pathway in transient expression assays. Expression of the 2.8 kb transcript correlates with reduced tumorigenicity in HeLa-fibroblast hybrids, suggesting a role in tumor suppression. We now report the detection of ST5 proteins in cellular extracts, demonstrate specific expression of p70 in non-tumorigenic HeLa-fibroblast hybrids, extend the correlation between p70 expression and cellular morphology to a wide variety of cell lines, and provide direct evidence that p70 can effect changes in cell growth and morphology. ST5 proteins were identified in extracts of human, mouse and simian epithelial cells and fibroblasts, but were absent from lymphoid cells. Transfection of the 2.8 kb cDNA into a p70-negative mouse fibroblast line yielded stable transfectants with a flattened, less refractile morphology relative to controls. The p70 expressing clones had initial growth rates similar to those of control cells but their saturation density was reduced threefold, suggesting a restoration of contact-regulated growth. In conjunction with previous findings, these results suggest that ST5 proteins participate directly in events affecting cytoskeletal organization and tumorigenicity.

A vaccinia virus late transcription factor with biochemical and molecular identity to a human cellular protein.

A factor designated VLTF-X is required to support vaccinia virus late transcription in vitro. It has been found that a late promoter DNA binding activity cochromatographs and cosediments with VLTF-X activity. Current experiments show that VLTF-X activity is present in a variety of uninfected mammalian cell types and is indistinguishable from that recovered from infected cells based upon several criteria. VLTF-X activity from both sources displays the same purification profile over phosphocellulose and DNA affinity resins and has the same sedimentation coefficient. In addition, the factors purified from both infected and uninfected cells form protein-DNA complexes of identical electrophoretic mobility in the presence of vaccinia virus late promoter-containing DNA. The affinity of these factors for the late promoter probes is identical and late promoter-specific based on competition experiments. Moreover, VLTF-X purified from both sources bound to late promoter-containing DNA in the presence or absence of MgCl2 and ATP and formed complexes resistant to heat inactivation. These experiments offer proof that vaccinia virus factor VLTF-X is a host cell protein that supports transcription of the viral late genes.

Activation of extracellular signal-regulated kinase 2 by a novel Abl-binding protein, ST5.

The human ST5 gene encodes three proteins with predicted molecular masses of 126, 82, and 70 kDa. These widely expressed proteins share a C-terminal region that bears significant sequence homology to a group of GDP/GTP exchange proteins for the Rab3 family of small GTP binding proteins. The N-terminal region of the largest ST5 protein, p126, contains two proline-rich sequences, PR1 and PR2, with consensus motifs similar to Src homology 3 (SH3) binding regions and to mitogen-activated protein kinase (MAPK) phosphorylation sites. Based on these properties, we sought to investigate the activity of ST5 proteins in signal transduction pathways. In vitro, p126 displayed preferential binding to c-Abl SH3, as compared with other SH3 domains. This interaction was mediated by the PR2 sequence. In vivo, expression of p126, but not p82 or p70, activated MAPK/ERK2 in response to EGF in COS-7 cells. Expression of c-Abl with p126 greatly enhanced this activity. Deletion of PR1 blocked the ability of p126 to activate ERK2. Deletion of PR2 did not affect the basal activity, but blocked the stimulatory effect of c-Abl. Whereas p82 expression had no effect on ERK2 activation by p126, p70 completely abrogated this activity. These observations suggest that ST5 can function as a signaling protein and can provide a link between c-Abl and ERK2.

A vaccinia virus late transcription factor copurifies with a factor that binds to a viral late promoter and is complemented by extracts from uninfected HeLa cells.

We have previously described a vaccinia virus late transcription factor, VLTF-X, which we found to be present in cells at early and late times in infection. In this study, transcription complementation assays were used to demonstrate that VLTF-X activity is also present in virion extracts and in the cytoplasm of uninfected HeLa cells. Mobility shift assays performed on various VLTF-X preparations revealed that a late promoter DNA-binding activity cochromatographed and cosedimented with VLTF-X activity. Competition experiments demonstrated that this binding was specific for the late promoter region of the probe and that late transcription was dramatically reduced by an oligonucleotide that blocked factor-DNA complex formation but was only minimally affected by an oligonucleotide that did not inhibit complex formation. These results suggest that a cellular factor may participate in vaccinia virus late transcription. These findings also confirm the requirement for VLTF-X and distinguish it from any of the previously described vaccinia virus late transcription factors, which have all been mapped to the viral genome. Finally, these studies also suggest that the biochemical role for VLTF-X may be in late promoter recognition.

KIT mutation portends poor prognosis in gastrointestinal stromal/smooth muscle tumors.

Gastrointestinal stromal/smooth muscle tumors (GISTs) are uncommon neoplasms for which current criteria for the diagnosis of malignancy (location, size, and mitotic index) do not always reliably predict patient outcome. Recently, mutation of KIT oncogene exon 11 has been observed in some of these tumors, but the relationship between mutation and clinical outcome has not yet been determined. DNA was obtained from the formalin-fixed, paraffin-embedded tissue of 35 gastric GISTs. A segment of exon 11 was amplified by PCR and sequenced on an ABI 377 sequencer. The relationship between the presence or absence of mutation, tumor size, and mitotic index was investigated using correlation analysis, and the relationship between mutation and outcome was investigated using Kaplan-Meier plots, the Cox-Mantel statistic, and the Cox regression model. Exon 11 deletion mutations were identified in 10 cases, and point mutations were identified in an additional 3 cases; 22 cases demonstrated no KIT mutations. KIT mutation was associated with decreased survival (p = 0.001), with fewer than 30% of patients surviving more than 3 years, compared with over 65% survival for patients whose tumors did not bear the mutation. KIT mutation did not correlate with either the mitotic index or the tumor size. In conclusion, KIT mutation is associated with poor prognosis in patients with gastrointestinal stromal tumors. Whether the KIT mutation will prove to be an independent prognostic factor awaits the completion of larger studies.

The A2L intermediate gene product is required for in vitro transcription from a vaccinia virus late promoter.

Previously, the in vitro late transcription system of vaccinia virus was resolved into four components: the 17- and 30-kDa products of the A1L and G8R intermediate genes, respectively, the viral DNA-dependent RNA polymerase, and an unmapped factor sedimenting at 32 to 38 kDa. Another protein, the 26-kDa product of the A2L open reading frame was predicted to be a late transcription factor on the basis of a transient-expression assay but was not recognized as being necessary for transcriptional activity in vitro. We now report that both the unmapped factor and the 26-kDa protein are required for transcription from a vaccinia virus late promoter in vitro. Since the 26-kDa protein has now been shown to be a trans-activator of late transcription and it is the product of a known gene, we suggest that it be designated VLTF-3.

Assembly of in vitro synthesized large subunits into ribulose-bisphosphate carboxylase/oxygenase. Formation and discharge of an L8-like species.

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) from higher plants consists of eight approximately 53-kDa large subunits and eight approximately 14-kDa small subunits. Cytosolic ribosomes synthesize the small subunits as precursors, which enter the chloroplast, undergo proteolytic processing, and assemble with large subunits. Large subunits, synthesized in the chloroplast, first form a complex with the chloroplast chaperonin 60 (Cpn60(14)). In the presence of ATP, large subunits dissociate from Cpn60(14) and assemble into Rubisco. We now describe partial characterization of a new species, Z, containing radiotracer-labeled, newly synthesized pea Rubisco large subunits. Rubisco assembly occurs in low salt in the presence of small subunits and ATP. As with Rubisco assembly, the formation of Z is ATP-dependent and is inhibited by high chloride. Once formed, Z is stable except in high chloride. Z does not appear to interact directly with small subunits. However, after Z formation, Rubisco assembly occurs in an ATP-independent reaction that requires KCl and small subunits. These results are consistent with the hypothesis that Z is a large subunit containing structure that can contribute large subunits to Rubisco under appropriate conditions. Z shares some physical characteristics with reported cyanobacterial L8 core particles. However, formation of Rubisco from Z in the absence of ATP and the presence of small subunits appears to require conditions that otherwise destabilize Z.

Assembly of in Vitro-Synthesized Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase Is Sensitive to CI-, Requires ATP, and Does Not Proceed When Large Subunits Are Synthesized at Temperatures [greater than or equal to]32[deg]C.

In higher plants, ribulose bisphosphate carboxylase/oxygenase (Rubisco) consists of eight large "L" subunits, synthesized in chloroplasts, and eight small "S" subunits, synthesized as precursors in the cytosol. Assembly of these into holoenzyme occurs in the chloroplast stroma after import and processing of the S subunits. A chloroplast chaperonin interacts with the L subunits, which dissociate from the chaperonin before they assemble into holoenzyme. Our laboratory has reported L subunit assembly into Rubisco in chloroplast extracts after protein synthesis in leaves, intact chloroplasts, and most recently in membrane-free chloroplast extracts. We report here that the incorporation of in vitro-synthesized L subunits into holoenzyme depends on the conditions of L subunit synthesis. Rubisco assembly did not occur after L subunit synthesis at 160 mM KCI. When L subunit synthesis occurred at approximately 70 mM KCI, assembly depended on the temperature at which L subunit synthesis took place. These phenomena were the result of postsynthetic events taking place during incubation for protein synthesis. We separated these events from protein synthesis by lowering the temperature during protein synthesis. Lower temperatures supported the synthesis of full-length Rubisco L subunits. The assembly of these completed L subunits into Rubisco required intervening incubation with ATP, before addition of S subunits. ATP treatment mobilized L subunits from a complex with the chloroplast chaperonin 60 oligomer. Addition of 130 mM KCI at the beginning of the intervening incubation with ATP blocked the incorporation of L subunits into Rubisco. The inhibitory effect of high KCI was due to CI- and came after association of newly synthesized L subunits with chaperonin 60, but before S subunit addition. It is interesting that L subunits synthesized at [greater than or equal to]32[deg]C failed to assemble into Rubisco under any conditions. These results agree with previous results obtained in this laboratory using newly synthesized L subunits made in intact chloroplasts. They also show that assembly of in vitro-synthesized L subunits into Rubisco requires ATP, that CI- inhibits Rubisco assembly, and that synthesis temperature affects subsequent assembly competence of L subunits.