Retraction Note: MiR-195-5p inhibits the cell migration and invasion of cervical carcinoma through suppressing ARL2.

The article "MiR-195-5p inhibits the cell migration and invasion of cervical carcinoma through suppressing ARL2", by S.-S. Pan, H.-E. Zhou, H.-Y. Yu, L.-H. Xu, published in Eur Rev Med Pharmacol Sci 2019; 23 (24): 10664-10671-DOI: 10.26355/eurrev_201912_19764-PMID: 31858533 has been retracted by the Authors for the following reasons: The authors found some inaccuracies in the research due to the number of experiments, as well as problems in the editing process of pictures. These errors may mislead readers and affect scientific research in this field. Figures 2D and 5D have also been questioned on PubPeer in April 2023. https://www.europeanreview.org/article/19764 This manuscript has been withdrawn. The Publisher apologizes for any inconvenience this may cause.

[Active screening of intestinal carbapenem-resistant Enterobacteriaceae in high-risk patients admitted to the hematology wards and its effect evaluation].

Objective: To evaluate the effect of intestinal carbapenem-resistant Enterobacteriaceae (CRE) active screening combined with enhanced intervention in the prevention and control of nosocomial infection in patients admitted to the hematological ward. Methods: Patients who were admitted to the Department of Hematology in a tertiary-care general hospital from March 1, 2017 to December 31, 2019 and underwent chemotherapy or immunosuppressive therapy comprised the intervention group. They were screened for intestinal CRE at least thrice. From December 1, 2016 to February 28, 2017, patients who underwent chemotherapy or immunosuppressive therapy without active intestinal CRE screening in the Department of Hematology formed the control group. Both the patient groups were monitored for CRE infection in real time. The χ(2) test was used to compare the changes in the CRE infection rate and mortality in high-risk patients before and after the active screening. Results: During the intervention period, the CRE colonization rate of patients was 16.46% (66/401) ; in terms of disease distribution, the colonization rate of acute leukemia was the highest 23.03% (26/113) . Of the 66 colonized patients, 27 (40.9%) patients were identified as positive for CRE at the first screening, 15 (22.7%) were identified at the time of the second screening, and the remaining 24 (36.4%) were identified at the third or subsequent screening; Carbapenem-resistant Klebsiella pneumoniae (CRPK) strains were dominant among the pathogens, accounting for 54.55% (36/66) . During the active screening period, the CRE infection rate (2.49%) and mortality rate (50.00%) of high-risk patients were significantly lower than those of the controls (11.30% and 69.23%, respectively) . The pathogens of 10 CRE infection patients during the intervention period were exactly the same as the previous active screening pathogens, and the coincidence rate was 100%. Conclusion: The CRE colonization rate was the highest in patients with acute leukemia who were admitted in the hematology wards. CRPK is the main pathogen of CRE colonization, infection, and death. Increasing the frequency of screening can significantly raise the positive rate of screening, Active screening can effectively reduce the incidence and subsequent mortality of CRE in high-risk patients admitted in the hematological wards. High coincidence rate between CRE screening positive pathogens and subsequent CRE infection pathogens. Intestinal CRE screening can serve as an indicator of CRE bloodstream infection in patients with hematological diseases as well as provide information for antibiotics therapy.

[Acute lymphoblastic leukemia complicated with cerebral venous thrombosis in 14 children].

Objective: To explore the clinical characteristics and management of childhood acute lymphoblastic leukemia (ALL) complicated with cerebral venous thrombosis (CVT). Methods: The clinical data of 14 ALL children complicated with CVT who were admitted to Department of Pediatrics of Sun Yat-sen Memorial Hospital and underwent chemotherapy from January 2011 to October 2019 were collected retrospectively. The clinical manifestations, coagulation function, imaging findings, treatment plan and prognosis of patients were analyzed. Results: CVT was diagnosed in 14 (2.8%, 14/505) cases, with a median age of 10 (3-14) years at onset, 11 cases occurred in the stage of induction remission, and the acute onsets were mainly characterized by convulsions (9 cases), consciousness disorders (6 cases) and headache (4 cases). Coagulation function test showed that, before the CVT, antithrombin Ⅲ activity was lower than 60% in 8 cases, D-dimer elevated on the day of onset in 8 cases. Arteriovenous angiography showed filling defects in single (9 cases) or multiple (5 cases) venous sinuses. The most common site of venous sinus enlargement was superior sagittal sinus (10 cases). Secondary cerebral hemorrhage was found in 5 cases. Anticoagulation therapy included combination of low-molecular-weight heparin (LMWH) and warfarin in 9 cases, sequential application of LMWH and warfarin in 2 cases, and LMWH alone in 3 cases. Patients accepted further asparaginase and no CVT recurrence or progression was found. Conclusions: The secondary coagulation dysfunction during induction remission chemotherapy is the major risk factor for CVT in ALL, which needs active monitoring and early prevention. Arteriovenous angiography can diagnose accurately, and the prognosis of anticoagulant therapy with LMWH and warfarin is optimistic.

MiR-195-5p inhibits the cell migration and invasion of cervical carcinoma through suppressing ARL2.

OBJECTIVE: MicroRNAs (miRNAs) have great effects on the progression of cervical cancer (CC). This study aimed to investigate the role of miR-195-5p in CC and to explain the regulatory mechanism between ARL2 and miR-195-5p. PATIENTS AND METHODS: Quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR) was used to detect miR-195-5p levels in CC tissues and cell lines. Transwell assays for cell migration and invasion were also performed. A luciferase reporter assay was used to detect the direct target of miR-195-5p. The protein levels of ARL2 were measured by Western blot analysis. RESULTS: In CC tissues and cell lines, miR-195-5p expression was decreased. Downregulation of miR-195-5p was associated with higher FIGO stage, deep stromal invasion, and lymph node metastasis. Moreover, over-expression of miR-195-5p inhibited cell migration and invasion in CC. Furthermore, it was observed that miR-195-5p directly targeted ARL2, which affected the suppressive effect of miR-195-5p in CC. CONCLUSIONS: MiR-195-5p inhibited cell migration and invasion in CC by suppressing ARL2 expression. The miR-195/ARL2 axis may provide a pathway for cell metastasis in CC.

Overexpressed miR-155 is associated with initial presentation and poor outcome in Chinese pediatric acute myeloid leukemia.

OBJECTIVE: Acute myeloid leukemia (AML) is the second leading leukemia in children. There is growing evidence that microRNAs (miRNAs) are crucial regulators involved in leukemogenesis. This study aimed to investigate the role of miR-155 in Chinese pediatric AML by evaluating its diagnostic and prognostic significance. PATIENTS AND METHODS: The expression of miR-155 and miR-25 in bone marrow specimens from 83 AML and 29 non-malignancies children were analyzed by TaqMan probe-based real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: The expression level of miR-155 was significantly higher in AML patients than in controls. Besides, a lowest miR-155 level was found in favorable prognosis group and t (15; 17)/M3 subgroup compared to the rest, while a higher level in C-Kit/FLT3-ITD mutation and relatively lower level existed in "Negative" mutation group. Moreover, miR-155 level was positively associated with the white blood cell (WBC) count, serum lactate dehydrogenase (LDH) and C-reaction protein (CRP) value in peripheral blood (PB), as well as miR-25/miR-196b expression levels. Survival analysis showed a statistically negative association with overall survival (OS) in the expression of miR-155 in chemotherapy group. CONCLUSIONS: These finding suggested that miR-155 expression cannot only be promising biomarker for the early detection of pediatric AML but also predict poor outcome. MiR-155 would be a novel biomarker for diagnosis, prognosis and therapy in pediatric AML.

Recovery of gastrointestinal function with thoracic epidural vs. systemic analgesia following gastrointestinal surgery.

The objective of this review was to systematically assess the effect of thoracic epidural analgesia (TEA) vs. systemic analgesia (SA) on the recovery of gastrointestinal (GI) function in patients following GI surgery. We performed a comprehensive literature search to identify randomized controlled trials of adult patients undergoing GI surgery, comparing the effect of two postoperative analgesia regimens. Patients postoperatively receiving local anesthesia-based TEA with or without opioids were compared to patients receiving opioid-based SA. The outcomes considered were times to GI function recovery, GI complications, and specific side effects. Twelve studies with 331 patients in the TEA group and 319 in the SA group were included. Compared to SA, TEA improved the GI recovery after GI procedures by shortening the time to first passage of flatus by 31.3 h, 95% confidence intervals (CIs): -33.2 to -29.4, P < 0.01; and shortening the time to first passage of stool by 24.1 h, 95% CIs: -27.2 to -20.9, P < 0.001. There was no difference between the groups in the incidence of anastomotic leakage and ileus. The occurrence of postoperative hypotension was relatively higher in the TEA group, risk ratio: 7.9, 95% CIs: 2.4 to 26.5, P = 0.001; other side effects (such as pruritus and vomiting) were similar in the two groups. There is evidence that TEA (compared to SA) improves the recovery of GI function after GI procedures without any increased risk of GI complications. To further confirm these effects, larger, better quality randomized controlled trials with standard outcome measurements are needed.

Proline-rich tyrosine kinase 2 and its phosphorylated form pY881 are novel prognostic markers for non-small-cell lung cancer progression and patients' overall survival.

BACKGROUND: Our previous study revealed that proline-rich tyrosine kinase 2 (Pyk2) is implicated in both anchorage-independent growth and anoikis resistance in lung cancer cells. This study aims to explore the expression and clinical significance of Pyk2 and its phosphorylated forms in non-small-cell lung cancer (NSCLC). METHODS: The mRNA and protein levels of Pyk2 or cancer stem cell markers (ALDH1a1, ABCG2 and Bmi-1) were either examined by reverse transcription-PCR or western blotting. An immunohistochemistry (IHC) assay was conducted to analyse the expression of Pyk2 and its phosphorylated forms in 128 NSCLC cases. RESULTS: The levels of Pyk2 mRNA, total protein, and its phosphorylated form pY881 were higher in lung cancer lesions than in the paired noncancerous tissues. The IHC analysis showed the levels of the Pyk2 and Pyk2[pY881] proteins were highly expressed in 70 (54.7%) and 77 (60.2%) cases, respectively. Both Pyk2 and Pyk2[pY881] were independent prognostic factors for NSCLC patients. The gain and loss study of Pyk2 function revealed that Pyk2 could upregulate the expression of ALDH1a1, ABCG2 and Bmi-1 and enhance the ability of colony formation in soft agar assay in A549 and H460 cells. CONCLUSION: Both Pyk2 and phosphorylated Pyk2[pY881] are potential prognostic factors and therapeutic targets for NSCLC.

Isolation and characterization of antagonistic bacteria against bacterial leaf spot of Euphorbia pulcherrima.

AIMS: To investigate the inhibition potential of leaf-associated bacteria against the pathogen of bacterial leaf spot of Euphorbia pulcherrima. METHODS AND RESULTS: Seven out of 200 bacterial strains were effective antagonists by in vitro screening and the two strains PAB241 and PAB242 significantly reduced the disease incidence and severity as foliar treatments of E. pulcherrima. The two effective strains, PAB241 and PAB242, were both identified as Bacillus amyloliquefaciens by a polyphasic approach including phenotypic feature, carbon source utilization profile, fatty acid methyl esters and analysis of 16S rRNA gene sequence. In addition, the suspensions of B. amyloliquefaciens PAB241 and PAB242 showed antibacterial activities against the pathogen of bacterial leaf spot of E. pulcherrima under different treatments. CONCLUSIONS: The leaf-associated bacteria, B. amyloliquefaciens PAB241 and PAB242, markedly inhibited the growth of X. axonopodis pv. poinsettiicola under different treatments and protected E. pulcherrima from pathogen infection in growth chamber conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that showed B. amyloliquefaciens from plant leaves was a potential bactericide against bacterial leaf spot of E. pulcherrima.

Antitumour and antimicrobial activities of endophytic streptomycetes from pharmaceutical plants in rainforest.

AIMS: The aim of this study was to screen antitumour and antimicrobial activities of endophytic actinomycetes isolated from pharmaceutical plants in rainforest in Yunnan province, China. METHODS AND RESULTS: Antitumour activity was studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and antimicrobial activity was determined by agar well diffusion method. The high bioactive endophytic isolates were identified and further investigated for the presence of polyketide synthases (PKS-I, PKS-II) and nonribosomal peptide synthetases (NRPS) sequences by specific amplification. The molecular identification confirmed that the 41 isolates showed significant activities were members of the genus Streptomyces. Among them, 31.7% of endophytic streptomycete cultures were cytotoxic against A549 cells, 29.3% against HL-60 cells, 85.4% against BEL-7404 cells, 90.2% against P388D1 cells, 65.9% were active against Escherichia coli, 24.4% against Staphylococcus aureus, 31.7% against Staphylococcus epidermidis, 12.2% against Candida albicans and no strain displayed antagonistic activity against Klebsiella pneumoniae. High frequencies of positive PCR amplification were obtained for PKS-I (34.1%), PKS-II (63.4%) and NRPS (61.0%) biosynthetic systems. CONCLUSIONS: Many endophytic streptomycetes isolated from pharmaceutical plants in rainforest possess remarkable and diverse antitumour and antimicrobial bioactivities. SIGNIFICANCE AND IMPACT OF THE STUDY: These endophytic streptomycetes are precious resources obtained from rainforests, and they could be a promising source for bioactive agents.

First Report of Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China.

In the spring of 2006, a new bacterial disease was noted in pear orchards near Hangzhou, Zhejiang Province, China. The disease caused severe blossom blast on pears (Pyrus pyrifolia; cv. Cuiguan). Early symptoms of the disease included blackening of the calyx end of developing fruit, blackening of blossom clusters while leaves of affected blossom clusters appeared normal, or death of clusters consisting of both blossoms and leaves. Later, tips of twigs turned dark brown and died. No bacterial ooze was observed. Twelve bacterial isolates were recovered from ten samples of buds and blossoms. Six isolates were selected for identification. They were similar to those of the reference strains of Pseudomonas syringae pv. syringae LMG5570 and LMG 2230 from Belgium in phenotypic tests on the basis of the Biolog Microbial Identification System (version 4.2; Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatographic analysis of fatty acid methyl esters (FAMEs) using the Microbial Identification System (MIDI Inc., Newark, DE) with aerobic bacterial library (TABA50), and electron microscopy (TEM, KYKY-1000B, Japan). All isolates tested were gram-negative, aerobic rods measuring 1.5 to 2.4 × 0.5 to 0.6 µm with 2 to 4 polar flagella. Fluorescent green diffusible pigment was produced on King's Medium B. Colonies were gray-white and slightly raised with smooth margins on nutrient agar. They produced levan on sucrose nutrient agar. A hypersensitive reaction was observed on tobacco cv. Benshi 24 h after inoculation. All isolates were identified as P. syringae pv. syringae with Biolog similarity index of 0.57 to 0.86 and FAME similarity index of 0.58 to 0.81. Identification as P. syringae pv. syringae was confirmed using 16S rDNA universal primers (2,3): 5'-AGA GTT TGA TCA TGG CTC AG-3' forward primer, 5'-ACG GTT ACC TTG TTA CGA CTT-3' reverse primer. The PCR fragments of the three isolates were sequenced and compared with sequences in GenBank. They had 99% similiarity with P. syringae pv. syringae 16S rRNA gene strain NCPPB 3869. Koch's postulates were conducted on buds of the original pear cultivar growing in pots and detached pear blossoms in flasks by spray inoculation with cell suspensions containing 108 CFU/ml of the six isolates at 18 to 22°C with two replications. The bacteria induced symptoms on buds and blossoms similar to those observed in the field. The bacterium was reisolated from symptomatic pear buds and internal ovary tissues. P. syringae pv. syringae was first reported in England as the cause of pear blossom blast in 1914 (1). After searching all the Chinese agricultural databases and major journals (National Knowledge Infrastructure database, Vip Chinese periodical database, Chinese wanfang database, China InfoBank, Scientia Agricultura Sinica, Acta Phytopathologica Sinica, Acta Phytophylacica Sinica, and Journal of Fruit Science), to our knowledge, this is the first report of pear blossom blast caused by P. syringae pv. syringae in China. The disease cycle on pear trees and the control strategies in the regions are being further studied. References: (1) B. P. Barker et al. Ann. Appl. Biol. 1:85, 1914. (2) U. Edward et al. Nucleic Acids Res. 17:7843,1989. (3) B. Li et al. J. Phytopathol. 34:141, 2006.

Concordant expression of proto-oncogene promyelocytic leukemia and major histocompatibility antigen HLA class I in human hepatocellular carcinoma.

Many malignant cancer cells downregulate human leukocyte antigen (HLA) class I antigen expression to evade T cell recognition. However, hepatocellular carcinoma (HCC) is exceptional to the general findings in cancer cells, and the mechanisms for its upregulation remain unclear. It has been reported that promyelocytic leukemia (PML) proto-oncogene controls the transcription of multiple class I antigen presentation genes in murine cancer cells. To find out the functional role of PML gene on the increased HLA class I antigen expression in HCC cells, we analyzed the expression of proto-oncogene PML and multiple class I antigen presentation genes in HCC specimens obtained in China. The results showed concordant changes of proto-oncogene PML and cell surface HLA-A expression in 44 paraffin-embedded HCC tissues. Furthermore, co-upregulated expression of PML genes and class I antigen presentation genes could be detected in 9 of 15 fresh HCC tissues by reverse transcription polymerase chain reaction (RT-PCR). In addition, studies using HCC cell lines showed that increased expression of HLA class I molecules paralleled with PML upregulation were detected in QGY-7701 HCC cell line with RT-PCR, western blot, and flow cytometry, and that the overexpression of exogenous PML in a low-expression class I cell line BEL-7405 could induce the expression of multiple class I antigen-presenting molecule genes and slightly but significantly increase the expression of cell surface HLA class I molecules. In conclusion, the expression of proto-oncogene PML and HLA class I molecules were concordantly upregulated and the expression of PML gene might be one of the mechanisms that leads to the increased expression of class I antigen in HCC.

Marrow graft rejection by repeated transfusions of allogeneic donor spleen cells.

Many hematological diseases require long-term transfusion support, which causes production of donor-reactive antibodies in sensitized recipients. Sensitized patients are at an increased risk for graft rejection when they undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we established a highly sensitized murine model to investigate the mechanism of donor graft rejection. After BALB/c mice were repeatedly transfused with allogeneic spleen cells from C57BL/6 mice, there was a significant increase in complement-dependent cytotoxicity in the serum of sensitized mice. For transplantation, 1 x 10(7) bone marrow cells (BMCs) from C57BL/6 mice were injected into lethally irradiated recipient BALB/c mice. Sensitized mice died between 12 and 15 days post-transplantation, while non-sensitized mice remained alive after 28 days. The hematopoietic recovery rate declined over time in sensitized recipients. The homing trace assay showed a rapid disappearance of donor BMCs in the spleen and bone marrow of sensitized recipients. In addition, the recipient cells and antibodies in the sensitized serum were capable of inducing high level of cell- and complement-mediated cytotoxicity to the donor graft. Our finding may explain the impaired hematopoietic stem cell homing and poor hematopoietic engraftment observed in highly sensitized allo-HSCT patients.

Formation of MTBE-DNA adducts in mice measured with accelerator mass spectrometry.

Methyl tert-butyl ether (MTBE) is a gasoline oxygenate and antiknock additive substituting for lead alkyls currently in use worldwide. Previous studies have shown that MTBE at very high doses induces tumors in rodents. The aim of the present study was to examine directly the binding ability of MTBE onto DNA, demonstrating its potential genotoxicity. MTBE-DNA adducts and their decay kinetics in mice have been measured by using doubly 14C-labeled MTBE with an advanced, ultrasensitive technique: accelerator mass spectrometry (AMS). It was found that MTBE definitely formed adducts with DNA in mouse lung, liver, and kidney in a log/log linear dose-response relationship. The distribution sequence of DNA adducts in these tissues is: lung > liver > kidney. The level of MTBE-DNA adducts peaked at 12 h postadministration in the lung and peaked at 6 h postadministration in the liver. Then the adducts declined rapidly until 5 days postadministration and thereafter declined much more slowly. To our knowledge, this is the first report on DNA adduction with MTBE in vivo. The mechanism of the formation of MTBE-DNA adducts also is discussed.

[Transplantation of cultured human keratinocyte on collagen sponge].

OBJECTIVE: To investigate the skin regeneration using cultured human keratinocytes with collagen sponge transplanted into thickness wound of nude mice. METHODS: Human foreskin from foreskin ectomy procedures was detached with 0.5% Dispase II. Epidermis sheets were separated from dermis and digested with 0.05% Trypsin into single cell suspension. Keratinocytes were cultured and seeded into collagen sponge during logarithmic growth phase. After 3 days, the keratinocytes-collagen sponge were grafted on full thickness wound of nude mice, compared with simple collagen sponge without keratinocytes. The histological, immunohistochemical examination and electron microscopy were detected. RESULTS: After the epidermal substitute was grafted onto wound, the human keratinocytes were able to further proliferate and differentiate and develop into new epithelia. Compared with the control group, the wound healed earlier and contracted less, epithelia matured earlier, and the collagen fiber was less beneath epithelia. CONCLUSION: Keratinocytes can grow on collagen sponge and migrate onto wound to develop into stratified epithelia and inhibit wound contract. The keratinocyte graft can be used to repair skin defect.

Detection of head and neck squamous cell carcinoma among exfoliated oral mucosal cells by microsatellite analysis.

Prompt detection of head and neck squamous cell carcinoma (HNSCC) is vital to successful patient management. In this feasibility study, we used microsatellite analysis to detect tumor-specific genetic alterations in exfoliated oral mucosal cell samples from patients with known cancer. Exfoliated mucosal cells in pretreatment oral rinse and swab samples were collected from 44 HNSCC patients and from 43 healthy control subjects (20 nonsmokers and 23 smokers). We tested a panel of 23 informative microsatellite markers to assay DNA from the matched lymphocyte, tumor (from cancer cases), and oral test samples. Loss of heterozygosity or microsatellite instability of at least one marker was detected in 38 (86%) of 44 primary tumors. Identical alterations were found in the saliva samples in 35 of these 38 cases (92% of those with markers; 79% overall) including 12 of 13 cases with small primaries [stage Tt or Tx (occult primary)] and 4 of 4 cases of patients that had undergone prior radiation. Microsatellite instability was detectable in the saliva in 24 (96%) of 25 cases in which it was present in the tumor, and loss of heterozygosity was identified in the test sample in 19 (61%) of 31 cases. No microsatellite alterations were detected in any of the samples from the healthy control subjects. This approach must now be refined and validated for the detection of clinically occult disease. Microsatellite analysis of oral samples may then become a valuable method for detecting and monitoring HNSCC.

The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Involvement of death receptor-related signaling pathways.

The focal adhesion kinase (FAK) is a mediator of cell-extracellular matrix signaling events and is overexpressed in tumor cells. In order to rapidly down-regulate FAK function in normal and transformed mammary cells, we have used adenoviral gene transduction of the carboxyl-terminal domain of FAK (FAK-CD). Transduction of adenovirus containing FAK-CD in breast cancer cells caused loss of adhesion, degradation of p125(FAK), and induced apoptosis. Furthermore, breast tumor cells that were viable without matrix attachment also underwent apoptosis upon interruption of FAK function, demonstrating that FAK is a survival signal in breast tumor cells even in the absence of matrix signaling. In addition, both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain and caspase-8, suggesting that a death receptor-mediated apoptotic pathway is involved. Finally, FAK-CD had no effect on adhesion or viability in normal mammary cells, despite the loss of tyrosine phosphorylation of p125(FAK). These results indicate that FAK-mediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway.

Engineering of a mini-trichosanthin that has lower antigenicity by deleting its C-terminal amino acid residues.

Trichosanthin is a ribosome-inactivating protein that possesses antitumor and antiviral activities. Clinical trials of trichosanthin on AIDS patients, however, elicit anaphylactic reactions. To reduce the antigenicity of trichosanthin as a drug while preserving its biological activity, the C-terminal domain (residues 203 to 247), which contains a putative antigenic site, was systemically deleted. We have found that the minimum length of trichosanthin that can fold into an active conformation is residue 1 to 240. The mini-trichosanthin (C7) generated by deleting the last seven C-terminal amino acid residues has 2.7-fold decrease in antigenicity, 10-fold reduction in in vitro ribosome-inactivation activity, and in vivo cytotoxicity toward K562 cells, and 2-fold reduction in abortificient activity. Structural analyses of C7 indicate decrease in the helix content, increased exposure of Trp192, and lower thermodynamic stability. The deletion of the C-terminal residues (Leu241 to Ala247) probably perturbs local structure of the C-terminal antigenic epitope that results in the decrease in antigenicity and activities of C7.

Site-directed polyethylene glycol modification of trichosanthin: effects on its biological activities, pharmacokinetics, and antigenicity.

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modified with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antigenic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sites were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential antigenic determinants, hence masking the antigenic region and prevent binding of antibodies specific to this site. In general, mutation did not bring about significant changes in ribosome-inactivating activity, cytotoxicity, and abortifacient activity of TCS. However, the in vitro activities of PEG modified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activity 50% less than that of nTCS. Pharmacokinetics study indicated that all three PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an IgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylation) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.

Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer.

BACKGROUND: Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of non-small-cell lung cancer (NSCLC). We have recently demonstrated that polymerase chain reaction (PCR)-based molecular methods are more sensitive than cytologic analysis in diagnosing bladder cancer. In this study, we examined whether molecular assays could identify cancer cells in bronchoalveolar lavage (BAL) fluid. METHODS: Tumor-specific oncogene mutations, CpG-island methylation status, and microsatellite alterations in the DNA of cells in BAL fluid from 50 consecutive patients with resectable (stages I through IIIa) NSCLC were assessed by use of four PCR-based techniques. RESULTS: Of 50 tumors, 28 contained a p53 mutation, and the identical mutation was detected with a plaque hybridization assay in the BAL fluid of 39% (11 of 28) of the corresponding patients. Eight of 19 adenocarcinomas contained a K-ras mutation, and the identical mutation was detected with a mutation ligation assay in the BAL fluid of 50% (four of eight) of the corresponding patients. The p16 gene was methylated in 19 of 50 tumors, and methylated p16 alleles were detected in the BAL fluid of 63% (12 of 19) of the corresponding patients. Microsatellite instability in at least one marker was detected with a panel of 15 markers frequently altered in NSCLC in 23 of 50 tumors; the identical alteration was detected in the BAL fluid of 14% (three of 22) of the corresponding patients. When all four techniques were used, mutations or microsatellite instability was detected in the paired BAL fluid of 23 (53%) of the 43 patients with tumors carrying a genetic alteration. CONCLUSION: Although still limited by sensitivity, molecular diagnostic strategies can detect the presence of neoplastic cells in the proximal airway of patients with surgically resectable NSCLC.

The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells.

Focal adhesion kinase (FAK) is a tyrosine kinase that is linked to signaling pathways between cells and their extracellular matrix. An alternate transcript of the COOH-terminal region of the FAK gene, called FAK-related nonkinase, has been shown to act as an inhibitor of FAK in chicken embryo fibroblasts. We have designed an analogous segment of human FAK, FAK COOH-terminal domain (FAK-CD), and transfected this construct into human tumor cells. Expression of FAK-CD inhibited cell growth in BT474 human breast cancer cells and C8161 human melanoma cells. To characterize the nature of growth inhibition, we developed an inducible system of FAK-CD expression and demonstrated that the induced FAK-CD protein localized to focal adhesions, causing cellular rounding, an irreversible loss of adhesion, and subsequent cell death. In addition, expression of FAK-CD reduced tyrosine phosphorylation of FAK, suggesting that FAK-CD may be a potent inhibitor of FAK in human tumor cells.