Analytical polyphosphate extraction from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, inorganic polyphosphate is analyzed by polyphosphate extraction and subsequent quantification. Recently, we developed a method for polyphosphate quantification, and length determination of short chain polyphosphate. However, the lack of a simple, optimized and validated method for analytical polyphosphate extraction has both hindered the advance in this research field, and prevented comparability of results between laboratories. Hence, the goal of this study was to develop an analytical method for polyphosphate extraction from S. cerevisiae. Several literature methods were compared with special attention to omission of polyphosphate precipitation steps, because these work neither at low polyphosphate concentrations nor quantitatively. The best literature protocol, which takes 5.5 h and requires five reaction tubes per sample, was optimized here in regards to the amount of extracted polyphosphate and simplification of the work flow. The final protocol extracts 40 % more polyphosphate than the best literature method, takes only 30 min, requires just one reaction tube per sample, and is, therefore, proposed as the new gold standard for analytical polyphosphate extraction from S. cerevisiae. In combination with our recently published polyphosphate quantification method, total polyphosphate in S. cerevisiae can now be analyzed within 2 h.

Expression of FHIT, p16, p53 and EGFR as prognostic markers in thyroid tumors of uncertain malignant potential.

PURPOSE: Thyroid tumors of uncertain malignant potential (TT-UMP) constitute a relatively new diagnosis. The purpose of this study was to analyze the relationship between immunohistochemical panels, prognostic parameters and TT-UMP. METHODS: Group I was composed of patients diagnosed as differentiated thyroid carcinoma (DTC) and Group II of patients diagnosed as TT-UMP. The prognostic scores of patients were calculated using data according to the well-known prognostic scoring systems MACIS, AMES, AGES. Evaluations of antibodies were based on the presence of nuclear staining for p16 and p53, membranous and cytoplasmic staining for epidermal growth factor receptor (EGFR) and cytoplasmic staining for fragile histidine triad (FHIT). RESULTS: Statistically significant difference was noted (p< 0.05) between Group I and Group II according to MACIS and AMES. No statistical difference was found in terms of immunostaining between groups when stained with p16, p53 and FHIT. On the other hand, in Group II a moderate positive correlation was detected between MACIS and EGFR. CONCLUSION: According to our findings p53 was not important in tumor genesis at early stages in well-differentiated thyroid carcinomas and p16 loss of expression could be used as a finding to help in difficult microscopic diagnosis. TT-UMP is a gray zone of lesions requiring specific therapeutic procedures and postoperative follow-up. A positive correlation was detected between EGFR and TT-UMP, leading to assume that this situation could be used as a new tool in the follow-up of these patients in the future.

A continuous enzyme-coupled assay for triphosphohydrolase activity of HIV-1 restriction factor SAMHD1.

The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes. SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells. We describe here an enzyme-coupled assay for quantifying the activation, inhibition, and hydrolysis of dNTPs, nucleotide analogues, and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents.

Selective monitoring of the enzymatic activity of the tumor suppressor Fhit.

Cancer is a leading cause of death worldwide. Functional inactivation of tumor suppressor proteins, mainly by mutations in the corresponding genes, is a key event in cancer development. The fragile histidine triade protein (Fhit) is a tumor suppressor that is frequently affected in different cancer types. Fhit possesses diadenosine triphosphate hydrolase activity, but although reduction of its enzymatic activity appears to be important for exerting its tumor suppressor function, the regulation of Fhit activity is poorly understood. Here, we introduce a novel fluorogenic probe that is suited to selectively analyze the enzymatic activity of Fhit in extracts derived from human cells. This novel method will allow in-depth insight into the mechanisms involved in Fhit regulation in biologically relevant setups and, thus, into its role in the development of cancer.

Multilevel correlations in the biological phosphorus removal process: From bacterial enrichment to conductivity-based metabolic batch tests and polyphosphatase assays.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the preferential selection of active polyphosphate-accumulating organisms (PAO) in the underlying bacterial community continuum. Efficient management of the bacterial resource requires understanding of population dynamics as well as availability of bioanalytical methods for rapid and regular assessment of relative abundances of active PAOs and their glycogen-accumulating competitors (GAO). A systems approach was adopted here toward the investigation of multilevel correlations from the EBPR bioprocess to the bacterial community, metabolic, and enzymatic levels. Two anaerobic-aerobic sequencing-batch reactors were operated to enrich activated sludge in PAOs and GAOs affiliating with "Candidati Accumulibacter and Competibacter phosphates", respectively. Bacterial selection was optimized by dynamic control of the organic loading rate and the anaerobic contact time. The distinct core bacteriomes mainly comprised populations related to the classes Betaproteobacteria, Cytophagia, and Chloroflexi in the PAO enrichment and of Gammaproteobacteria, Alphaproteobacteria, Acidobacteria, and Sphingobacteria in the GAO enrichment. An anaerobic metabolic batch test based on electrical conductivity evolution and a polyphosphatase enzymatic assay were developed for rapid and low-cost assessment of the active PAO fraction and dephosphatation potential of activated sludge. Linear correlations were obtained between the PAO fraction, biomass specific rate of conductivity increase under anaerobic conditions, and polyphosphate-hydrolyzing activity of PAO/GAO mixtures. The correlations between PAO/GAO ratios, metabolic activities, and conductivity profiles were confirmed by simulations with a mathematical model developed in the aqueous geochemistry software PHREEQC.

Temperature influence on biological phosphorus removal induced by aerobic/extended-idle regime.

Previous researches have demonstrated that biological phosphorus removal (BPR) from wastewater could be driven by the aerobic/extended-idle (A/EI) regime. This study further investigated temperature effects on phosphorus removal performance in six A/EI sequencing batch reactors (SBRs) operated at temperatures ranging from 5 to 30 °C. The results showed that phosphorus removal efficiency increased with temperature increasing from 5 to 20 °C but slightly decreased when temperature continually increased to 30 °C. The highest phosphorus removal rate of 97.1 % was obtained at 20 °C. The biomass cultured at 20 °C contained more polyphosphate accumulating organisms (PAO) and less glycogen accumulating organisms (GAO) than that cultured at any other temperatures investigated. The mechanism studies revealed that temperature affected the transformations of glycogen and polyhydroxyalkanoates, and the activities of exopolyphosphatase and polyphosphate kinase activities. In addition, phosphorus removal performances of the A/EI and traditional anaerobic/oxic (A/O) SBRs were compared at 5 and 20 °C, respectively. The results showed the A/EI regime drove better phosphorus removal than the A/O regime at both 5 and 20 °C, and more PAO and less GAO abundances in the biomass might be the principal reason for the higher BPR in the A/EI SBRs as compared with the A/O SBRs.

Reduced FHIT expression is associated with mismatch repair deficient and high CpG island methylator phenotype colorectal cancer.

Colorectal cancer (CRC) is a heterogeneous disease and a major contributor to world cancer mortality rates. Molecular subtypes of CRC have become standards for CRC classification and have established prognostic potential. Here, we attempt to corroborate and provide further insight pertinent to the fragile histidine triad (FHIT) gene in microsatellite instable (MSI), microsatellite stable (MSS), and CpG island methylator phenotype (CIMP) CRC subtypes. We employed array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA) techniques to survey genomic aberrations in FHIT gene and their effects on FHIT protein expression using immunohistochemistry (IHC) in a CRC cohort. We further studied FHIT protein expression by IHC in a larger CRC cohort defined for its mismatch repair (MMR) protein expression and genomic methylation profiles. Our results show FHIT genomic deletions centered in exons 4 and 5 in most of MSI-CRC samples. Moreover, we confirmed the significant association of FHIT protein expression diminution (p=0.035) with MSI-CRC. In the larger cohort, reduced FHIT protein expression was significantly associated with CIMP-high subtype of CRC (p=0.009) and loss of PMS2 protein expression (p=0.017). We conclude that FHIT expression may be a valuable marker for CRC subtyping, and its diagnostic, prognostic, and therapeutic potential should be perused.

Correlation between LRIG1 and LRIG2 expressions and expression of 11 tumor markers, with special reference to tumor suppressors, in CIN and normal cervical epithelium.

OBJECTIVE: Novel biological markers LRIG1 and LRIG2 have been associated with favorable as well as poor prognosis, respectively, in different cancer types, including cervical cancer. The aim of this study was to investigate possible interactions between these proteins and other tumor markers, and as diagnostic adjuncts in CIN. METHODS: Cervical biopsies from 171 women, with normal epithelium, and low-grade and high-grade CIN were stained for LRIG1 and LRIG2, and 11 additional tumor markers. The tumor markers were chosen to be relevant in cervical neoplasms. Staining was evaluated semiquantitatively. RESULTS: Expression of LRIG1 and LRIG2 was found to correlate with increasing CIN grade, as well as with expression of tumor suppressor FHIT, independent of histological grade. In addition, tumor promoter LRIG2 expression correlated negatively with expression of tumor suppressor retinoblastoma protein and positively with IL-10. The latter correlation did not however remain after adjustment for CIN grade. p53 and p16 expressions correlated positively with LRIG1 expression in univariate analyses, but significance did not hold after adjustment for CIN grade. CONCLUSION: LRIG1 and LRIG2 expressions were seen in precancerous cervical epithelium and found to increase with increasing grade. There was an association between expression of these glycoproteins and FHIT tumor suppressor protein, independently of histological grade.

High-risk HPV infection and CIN grade correlates to the expression of c-myc, CD4+, FHIT, E-cadherin, Ki-67, and p16INK4a.

OBJECTIVE: : This study aimed to investigate correlations between a panel of biomarkers/tumor markers and high-risk (HR) human papillomavirus (HPV)-positive versus HR-HPV-negative cervical lesions. MATERIALS AND METHODS: : The study included 188 women who consecutively attended a colposcopy clinic because of PAP smears suggesting cervical intraepithelial neoplasia (CIN), and 40 women with normal vaginal cytology. Tissue microarray blocks were prepared from representative cervical cone or punch biopsies. Sections were stained for 12 biological markers, previously shown to be relevant in cervical neoplasms, and expression was correlated to the presence or absence of HR-HPV in cervical lesions. RESULTS: : No correlations between expression of biomarkers and HPV status were found in normal epithelium. Expression of c-myc, CD4, Ki-67, and p16 correlated significantly to HR-HPV-infected epithelium compared with HR-HPV-negative epithelium. When adjustment was made for CIN grade, only the expression of Ki-67 correlated significantly with HPV status and CIN grade. Human papillomavirus status was stratified to normal epithelium, low-grade CIN, and high-grade CIN. Fragile histidine triad (FHIT), E-cadherin, Rb, Ki-67, and p16 expression was significantly increased in HPV-positive tissue by increasing CIN grade. No correlation to tumor marker expression was observed in the HPV-negative tissue. CONCLUSIONS: : This study described correlations, previously not investigated, between HPV status and tumor marker expression, that is, E-cadherin, Rb, and fragile histidine triad. Surprisingly, p16 was not, although Ki-67 expression was, independently correlated to HPV positivity. The results of this study suggest that p16 instead correlates independently with increasing CIN grade.

Aberrant expression of DNA damage response proteins is associated with breast cancer subtype and clinical features.

Landmark studies of the status of DNA damage checkpoints and associated repair functions in preneoplastic and neoplastic cells has focused attention on importance of these pathways in cancer development, and inhibitors of repair pathways are in clinical trials for treatment of triple negative breast cancer. Cancer heterogeneity suggests that specific cancer subtypes will have distinct mechanisms of DNA damage survival, dependent on biological context. In this study, status of DNA damage response (DDR)-associated proteins was examined in breast cancer subtypes in association with clinical features; 479 breast cancers were examined for expression of DDR proteins γH2AX, BRCA1, pChk2, and p53, DNA damage-sensitive tumor suppressors Fhit and Wwox, and Wwox-interacting proteins Ap2α, Ap2γ, ErbB4, and correlations among proteins, tumor subtypes, and clinical features were assessed. In a multivariable model, triple negative cancers showed significantly reduced Fhit and Wwox, increased p53 and Ap2γ protein expression, and were significantly more likely than other subtype tumors to exhibit aberrant expression of two or more DDR-associated proteins. Disease-free survival was associated with subtype, Fhit and membrane ErbB4 expression level and aberrant expression of multiple DDR-associated proteins. These results suggest that definition of specific DNA repair and checkpoint defects in subgroups of triple negative cancer might identify new treatment targets. Expression of Wwox and its interactor, ErbB4, was highly significantly reduced in metastatic tissues vs. matched primary tissues, suggesting that Wwox signal pathway loss contributes to lymph node metastasis, perhaps by allowing survival of tumor cells that have detached from basement membranes, as proposed for the role of Wwox in ovarian cancer spread.

Clinicopathological significance and prognostic value of leukemia-related protein 16 expression in invasive ductal breast carcinoma.

To explore the expression of leukemia-related protein 16 (LRP16) in invasive ductal breast carcinoma and analyze its correlation with clinicopathological feature and prognosis, immunohistochemistry was performed on 100 cases of invasive ductal breast carcinoma. Medical records were reviewed and clinicopathological analysis was performed. Leukemia-related protein 16 expression was detected in 33 of 100 cases (33%) of the invasive ductal breast carcinoma. Expression of LRP16 in carcinoma was obviously higher than that in normal breast tissue. LRP16 protein expression was found in 27.6% (21/76) of carcinoma at stage I and II, and 50.0% (12/24) of carcinoma at stage III and IV. LRP16 expression was found correlative with metastasis in the axillary lymph node (P = 0.001), stage (P = 0.042), estrogen receptor (ER) expression (P = 0.001), fragile histidine triad (FHIT) expression (P = 0.015) and CD133 expression (P = 0.038), but not with grade (P = 0.543), tumor size (P = 0.263), age (P = 0.840), menopause (P = 0.701) and HER-2 gene amplification (P = 0.463). The difference of the mean disease free survival (DFS) time between cancer patients with LRP16 expression (43.7 months) and those without (77.7 months) was statistically significant (Log rank = 9.989, P = 0.002). The difference of the mean overall survival (OS) time between cancer patients with LRP16 expression (50.0 months) and those without (120.0 months) was statistically significant (Log rank = 9.977, P = 0.002). Our finding suggests that expression of LRP16 protein is correlated with the stage, metastasis, prognosis and expression of ER, progesterone receptor, Ki-67, CD133 and FHIT in invasive ductal breast carcinoma.

Use of multiple biological markers in radiotherapy-treated head and neck cancer.

OBJECTIVE: Management of patients with head and neck squamous cell carcinoma is often based on clinical parameters, with little appreciation of the underlying tumour biology. Single biological marker studies fail to acknowledge the complexity of these tumours. Our aim was to define a profile of biological markers associated with outcome. DESIGN: This retrospective study involved consecutive patients with oropharyngeal squamous cell carcinoma treated with primary radiotherapy between 1996 and 2001. Pre-treatment biopsies were used to study the immunohistochemical expression of nine biological markers. Markers were chosen to reflect biologically relevant pathways. RESULTS: Following analysis of nine markers, a profile of two markers was derived (carbonic anhydrase 9 and major vault protein), the co-expression of which conferred a significantly poor probability of locoregional control. The prognostic effect of these biomarkers in combination was greater than their effect individually. CONCLUSION: Biomarker profiles can be established which highlight large differences in locoregional control. Identifying tumours that express both carbonic anhydrase 9 and major vault protein may facilitate patient selection for more aggressive treatment.

Inorganic polyphosphate induces osteoblastic differentiation.

Inorganic polyphosphate [Poly(P)] is especially prevalent in osteoblasts. We tested the hypothesis that Poly(P) stimulates osteoblastic differentiation and polyphosphate metabolism for bone formation. The osteoblast-like cell line, MC 3T3-E1, was cultured with Poly(P), and gene expression was evaluated by real-time reverse-transcription polymerase chain-reaction. Phosphatase activity and extracellular matrix mineralization were also determined. The role of Poly(P) was assessed in a beagle dog alveolar bone regeneration model. Poly(P) increased osteocalcin, osterix, bone sialoprotein, and tissue non-specific alkaline phosphatase gene expression, with a high level of end-polyphosphatase activity, resulting in low-chain-length Poly(P), inorganic pyrophosphate, and inorganic phosphate production. MC3T3-E1 cells differentiated into mature osteoblasts and showed expression of ectonucleotide pyrophosphatase phosphodiesterase 1, while mouse progressive ankylosis gene expression remained unchanged. Promotion of alveolar bone regeneration was observed in Poly(P)-treated beagle dogs. These findings suggest that Poly(P) induces osteoblastic differentiation and bone mineralization, and acts as a resource for mineralization.

Intramitochondrial calcium regulation by the FHIT gene product sensitizes to apoptosis.

Despite the growing interest in the Fhit tumor suppressor protein, frequently deleted in human cancers, the mechanism of its powerful proapoptotic activity has remained elusive. We here demonstrate that Fhit sensitizes the low-affinity Ca(2+) transporters of mitochondria, enhancing Ca(2+) uptake into the organelle both in intact and in permabilized cells, and potentiating the effect of apoptotic agents. This effect can be attributed to the fraction of Fhit sorted to mitochondria, as a fully mitochondrial Fhit (a chimeric protein including a mitochondrial targeting sequence) retains the Ca(2+) signaling properties of Fhit and the proapoptotic activity of the native protein (whereas the effects on the cell cycle are lost). Thus, the partial sorting of Fhit to mitochondria allows to finely tune the sensitivity of the organelle to the highly pleiomorphic Ca(2+) signals, synergizing with apoptotic challenges. This concept, and the identification of the molecular machinery, may provide ways to act on apoptotic cell death and its derangement in cancer.

Loss expression of active fragile sites genes associated with the severity of breast epithelial abnormalities.

BACKGROUND: WWOX and FHIT are two candidate tumor suppressor genes located in active fragile sites, the damage of which has been associated with the development of breast cancer. The association of the expression of these genes and the development of breast cancer has not been fully explored. We evaluated mRNA and protein expression of WWOX and FHIT in breast tissue with normal histological appearances, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive cancer to see if a progressive decline in expression was present. METHODS: Reverse transcription-polymerase chain reaction and Western blotting were used to evaluate the specimens for mRNA and protein expression, including 28 specimens with normal tissue, 28 specimens with atypical ductal hyperplasia, 33 specimens with ductal carcinoma in situ, and 51 specimens with invasive ductal carcinoma. RESULTS: Compared with in situ and invasive cancer specimens, both normal and atypical hyperplasia specimens had greater rates of detectable mRNA (WWOX rate ratio = 2.95, 95% CI 1.24 - 7.08; FHIT rate ratio = 4.58, 95% CI 1.82 - 11.81) and Western blotting detectable protein (WWOX rate ratio = 4.12, 95% CI 1.63 - 10.73; FHIT rate ratio = 3.76, 95% CI 1.44 - 10.06). For both proteins, differences between normal and atypical hyperplasia specimens and between in situ and invasive carcinoma specimens were explainable by chance (P > 0.05 for each analysis). Within each histological category, differences among fractions of specimens showed that FHIT and WWOX mRNA and protein expression were explainable by chance (P > 0.05 for each analysis). CONCLUSION: Expression of FHIT and WWOX decreases along with breast tissue progress from a normal histological appearance to atypical ductal hyperplasia, in situ cancer, and the final invasive cancer.

Mononuclear phagocytes in the blood of turtles characterized by ultrastructural and cytochemical analyses and by phagocytic activity.

Ultrastructural and cytochemical characteristics of mononuclear phagocyte cells in turtles are not well described in the literature, especially in Phrynops hilarii. Thus, the aim of this study was to evaluate these characteristics in the mononuclear phagocyte cells and their phagocytic activity "in vitro" using the turtle P. hilarii as an experimental animal model. The six turtles used in the study were observed in two seasons, spring and summer. Results showed that mononuclear phagocytes incubated only in diluted solution or with colloidal charcoal have cytoplasm phagolysosomes. The cells incubated with colloidal charcoal and further exposed to the cytochemical reaction for acid beta-glycerophosphatase, showed cytoplasm phagolysosomes filled by charcoal particles being digested and some positively stained lysosomes. Acid beta-glycerophosphatase positive reaction was present in lysosomes and inside the phagolysosomes, while acid cytidine 5-monophosphatase staining occurred in lysosome surroundings. A positive reaction for trimetaphosphatase was also found inside phagolysosomes. In conclusion, the presence of lysosomal enzymes like trimetaphosphatase and cytidine-5'-sodium monophosphate, in the circulating blood of P. hilarii indicate that mononuclear phagocytes participate in the phagocytic process by gathering many phagocytic cells and forming multinucleated giant cells, which probably have a role in the blood "clearance" process.

Clinicopathological significance of the fragile histidine triad transcription protein expression in laryngeal carcinogenesis.

The fragile histidine triad (FHIT), frequently lost in many cancers, was identified as a candidate tumor suppressor gene at chromosome 3p locus 14.2. Loss of the FHIT protein because of the alteration or loss of heterozygosity by genetic deletion occurs in a variety of epithelial tumors including head and neck cancer. However, the biological function of the FHIT protein is still unknown and its role in intrinsic cellular proliferation remains particularly controversial in preinvasive lesions and invasive tumors of the head and neck. To clarify the role of the FHIT protein in laryngeal squamous cell carcinoma (LSCC) and to examine whether the expression of FHIT could be a prognostic parameter for laryngeal carcinogenesis, we investigated the relationship between the expression of the FHIT protein, other tumor suppressor gene products (p53 and p16), the cellular proliferation marker (Ki-67) and the survival time of patients with LSCC. In our study, there were significant differences (p<0.05) in the expression of FHIT between low grade dysplasia and LSCC. Additionally, survival time analysis showed a significant correlation between the reduction of FHIT expression and the length of disease-free survival (p<0.05) in patients with T1-T2 N0 laryngeal carcinoma. However, we did not confirm a relationship between the expression of FHIT, the other tumor suppressor gene products (p53 and p16) or the cellular proliferation marker (Ki-67). In conclusion, we provided evidence that the reduction of FHIT levels may be a useful prognostic indicator for the clinical outcome of laryngeal SCC. Our findings indicated that FHIT utilizes a pathway independent of p53 and is involved in abnormal cell proliferation via the breakdown of G0-G1 arrest in the larynx and apoptosis during multistep carcinogenesis of the larynx.

Low expression of FHIT and PTEN correlates with malignancy of gastric carcinomas: tissue-array findings.

To clarify the roles of FHIT (fragile histidine triad) and PTEN (phosphatase and tensin homology deleted from human chromosome 10) expression in the genesis and progression of gastric cancers, we examined expression of FHIT and PTEN on tissue microarray containing gastric normal mucosa (n=49), adenoma (n=49), noncancerous mucosa adjacent to carcinoma (n=84) and carcinoma (n=249) by immunohistochemistry. Their expression was compared with clinicopathologic parameters of tumors, including expression of p53 and cysteine protease protein 32 as well as survival time of patients with carcinoma. The results showed expression of FHIT and PTEN were lower in gastric carcinoma than those in normal mucosa, noncancerous mucosa adjacent to carcinoma and adenoma of the stomach (P<0.05). FHIT and PTEN expression showed a significantly negative association with depth of invasion, lymphatic invasion, and lymph node metastasis, liver metastasis, and Union Internationale Contre le Cancer staging of gastric carcinoma (P<0.05). Intestinal-type gastric carcinomas highly expressed FHIT and PTEN protein, compared with diffuse-type ones (P<0.05). Expression of FHIT and PTEN were positively related with expression of p53 and cysteine protease protein 32 in gastric carcinoma (P<0.05), as well as favorable prognosis of the patients with the tumors (P<0.05). There was positive relationship between FHIT and PTEN expression in gastric carcinoma (P<0.05). It was suggested that down-regulated expression of FHIT and PTEN contributed to gastric carcinogenesis possibly by involving in the imbalance between apoptosis and proliferation of cells. Their altered expression underlay the molecular basis of invasion, metastasis, differentiation of gastric carcinoma.

Fhit protein expression in lung cancer studied by high-throughput tissue microarray.

AIM: To study the expression of Fhit protein in lung cancers and its potential relevance in the diagnosis and prognosis of lung cancers. METHODS: Tissue microarrays (TMA) and Immunohistochemistry (IHC) were performed in 321 cases of lung cancers. Fhit protein was tested by the S-P immunohistochemistry method upon tissue microarrays. RESULTS: In our TMA blocks comprising 321 cases of lung cancer, there were 253 (78.8 %) cases valid for Fhit protein assessment. Total loss or marked reduction of Fhit expression (-, +) were seen in 170 cases (67.1 %). Fhit protein loss in NSCLCs (non-small cell lung carcinoma was significantly lower than in SCLCs (small cell lung cancers) (p < 0.05), in most squamous cell carcinomas (85 out of 105, 81.0 %), and in a smaller portion of adenocarcinomas (53 out of 109, 48.6 %; p < 0.05). The loss or marked reduction of Fhit expression was more common in tumours occurring in smokers and male patients (133 out of 191, 69.6 %; 135 out of 189,71.4 %) as compared to nonsmokers and female patients (29 out of 62, 46.8 %; p < 0.005 and 29 out of 64, 45.3 %; p < 0.005). However, the expression of Fhit protein was not associated with histopathologic grading, clinical staging, lymph node metastasis or survival time. CONCLUSIONS: Our studies showed that the loss or reduced expression of Fhit, a tumour suppressor gene is a frequent occurrence in lung cancers, with variations amongst different histological subtypes. The loss or marked reduction of Fhit expression was more frequent in smokers. This study showed that tissue microarrays can be used efficiently for evaluation of the expression of certain tumor markers.

Enzymatic defects of the nsP2 proteins of Semliki Forest virus temperature-sensitive mutants.

We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24 degrees C and 35 degrees C. The protease domain mutants (ts4, ts6, and ts11) had reduced protease activity at 35 degrees C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.