Platelet activation via the collagen receptor GPVI is not altered in platelets from chronic myeloid leukaemia patients despite the presence of the constitutively phosphorylated adapter protein CrkL

In this study, we show that the adapter proteins CrkL and Cbl undergo increases in tyrosine phosphorylation and form an intracellular complex in platelets stimulated with the snake venom toxin convulxin, a selective agonist at the collagen receptor glycoprotein VI (GPVI). Constitutive tyrosine phosphorylation of CrkL has previously been reported in platelets from chronic myeloid leukaemia (CML) patients. This was confirmed in the present study, and shown to result in a weak constitutive association of CrkL with Cbl and a number of other unidentified tyrosine-phosphorylated proteins. There was no further increase in phosphorylation of CrkL in CML platelets in response to GPVI activation, whereas phosphorylation of Cbl and its association with CrkL were potentiated. In addition, this was accompanied by a small increase in p42/ 44 mapkinase (MAPK) activity in CML platelets. The functional consequence of the presence of constitutively phosphorylated proteins in CML platelets was investigated by measurement of aminophospholipid exposure and alpha-granule secretion. This revealed little alteration in the concentration-response curves for either in CML platelets stimulated via GPVI, although maximal levels of P-selectin were depressed. Despite the minimal effect on platelet activation in CML patients, we cannot exclude a role for CrkL or Cbl in signal transduction pathways stimulated via GPVI.     

人源性大肠癌自然致敏噬菌体抗体Fab呈现库的构建与筛选

目的构建大肠癌病人自然致敏淋巴结抗体 Fab 段噬菌体呈现库,初步筛选相关抗大肠癌抗体。方法取大肠癌病人转移淋巴结,提取淋巴结总 RNA,逆转录 PCR 扩增重链 Fd 和 k 轻链 cDNA。依次将 PCR 产物插入载体 pComb3的相应部位,构建人源性大肠癌噬菌体 Fab 基因库,并应用噬菌体表面呈现技术对该抗体库进行淘选及鉴定。结果所选2种 Ig 亚类的重链 Fd 片段、2种 k 轻链 cDNA 得到扩增。Fd 片段和 k 轻链均插入 pComb3的重组率为40%,Fab 噬菌体表达库容达1.48×10~6。经3轮淘选,抗体库得到约120倍的富集。3轮抗体库的点印记免疫染色均显示有 Fab 表达:酶联免疫吸附实验显示其与大肠癌抗原有结合活性。结论成功构建了大肠癌病人自然致敏淋巴结抗体 Fab 段噬菌体呈现库,利用噬菌体抗体库技术,可筛选相关抗大肠癌抗体。     

Aggregates in blood filter chambers used from the plasma donations of anti-D donors: evaluation for monoclonal antibody discovery using phage display

BackgroundRhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor’s donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display.Material and methodsHaemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count.ResultsOf 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity.DiscussionBFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display.     

Organotypic slice cultures of pancreatic ductal adenocarcinoma preserve the tumor microenvironment and provide a platform for drug response

Background

/Objective: The conventional models currently used to evaluate various anti-tumor therapeutic agents are not sufficient for representing human pancreatic ductal adenocarcinoma (PDA), which has a unique tumor microenvironment. We aimed to produce an organotypic slice culture model from human PDA that resembles the in vivo situation and to evaluate the responses of PDA slices to established cytotoxic drugs.

A procedure for measuring the contributions of intracellular and extracellular tyrosine pools to the rate of myocardial protein synthesis

To determine the relative contribution of intracellular and extracellular amino acid sources as precursors for protein synthesis, rat left atria were incubated in l-[³H]tyrosine at medium tyrosine concentrations ranging from 0.05 to 2.5 mm. Under conditions where protein synthesis was shown to be constant, tyrosine incorporation rates calculated from either the intracellular or extracellular tyrosine concentration. This behavior is consistent with a model which postulates that amino acids inside and outside the cell simultaneously supply a common precursor pool. A method is presented for determining the contributions of the intracellular and extracellular pools to the total protein synthesis rate. Analysis of tyrosine incorporation data according to this procedure yields a total incorporation rate of 1.14 ± 0.18 (s.e.m.) nmol tyrosine/mg protein/h, of which 61 ± 15% is attributed to a mixed intracellular source and 39 ± 7% to amino acids taken directly from the extracellular medium. During a 3-h incubation of the isolated atria protein synthesis and degradation rates were equal, and the oxygen consumption and ATP and creatine phosphate levels were similar to those of the beating, perfused heart.     

SLC22A8: An indicator for tumor immune microenvironment and prognosis of ccRCC from a comprehensive analysis of bioinformatics

Clear cell renal cell carcinoma (ccRCC) is one of the most common renal malignancies worldwide. SLC22A8 plays a key role in renal excretion of organic anions. However, its role in ccRCC remains unclear; therefore, this study aimed to elucidate the relationship between SLC22A8 and ccRCC. The The Cancer Genome Atlas-kidney renal clear cell carcinoma cohort was included in this study. The Wilcoxon signed-rank test and logistic regression were used to analyze the relationship between SLC22A8 expression and clinicopathological characteristics. Multifactorial analysis and Kaplan–Meier survival curves were adopted for correlation between SLC22A8 expression and clinicopathological parameters and overall survival. Utilizing the UALCAN database, the correlation of the expression levels of SLC22A8 DNA methylation in ccRCC was explored. Immunological characterization of SLC22A8 regarding the ccRCC tumor microenvironment was carried out by the single sample Gene Set Enrichment Analysis algorithm and the CIBERSORT algorithm. With the CellMiner database, the analysis of the association between SLC22A8 gene expression and drug sensitivity was further performed. Eventually, gene ontology and Kyoto Encyclopedia of Gene and Genome enrichment analyses were applied to identify the functional and signaling pathways involved in SLC22A8. SLC22A8 expression is associated with age, grade, stage, and tumor status. SLC22A8 protein expression levels, phosphorylated protein levels, and DNA methylation expression levels were lower in ccRCC tissues than in normal tissues, and low methylation levels predicted poor overall survival. Comprehensive analysis of tumor immune infiltration and the tumor microenvironment indicated a higher level of overall immunity in the SLC22A8 low expression group. Gene Enrichment Analysis results showed that low expression of SLC22A8 was associated with immune pathways, such as phagocytosis recognition and humoral immune response. SLC22A8 expression was significantly correlated with survival and immune infiltration in ccRCC and can be used as a prognostic biomarker for ccRCC.     

A rapid and simple procedure for dissociation of tumor tissue from the human colon

Summary A rapid procedure for dissociation of colon tumor tissue which combines enzymatic and mechanical methods is described. Using this method a cell suspension almost free of cell aggregates is obtained which is further characterized by high cell yield and excellent cell viability.Presented in part at the fourth symposium of the section of experimental cancer research (SEK) of the German Cancer Society (1987), J Cancer Res Clin Oncol 113:S8Dedicated to Professor Fritz Linder on the occasion of his 75^th birthday     

Evidence for a p27 tumor suppressive function independent of its role regulating cell proliferation in the prostate

Reduced p27 levels correlate with poor prognosis in a wide spectrum of human tumors and can accelerate tumorigenesis in mouse tissues. To determine whether p27 deficiency can accelerate tumorigenesis in tissues with inactive Rb and p53 pathways, we examined the effect of p27 status on prostate tumorigenesis in mice expressing simian virus 40 large T antigen (LT). In p27-deficient mice expressing LT, tumors progressed from high-grade prostatic intraepithelial neoplasia to poorly differentiated carcinoma at a greatly accelerated rate. p27 deficiency could not collaborate with a mutant of LT that fails to inactivate the Rb pathway alone. Furthermore, p27 deficiency does not increase the proliferation index, reduce the apoptotic index, or affect the expression of E2F-dependent genes in cells expressing LT at any stage of the disease. Expression of LT alone leads to maximal proliferation, but p27 deficiency still increases the amount of cyclin A and cyclin-dependent kinase 2-associated kinase activity in tissues. Interestingly, this model recapitulates an important feature of the human disease, specifically a high frequency of allelic loss of chromosome 16q, which is syntenic to mouse chromosome 8. Loss of heterozygosity may accelerate the inactivation of other tumor suppressors, such as E-cadherin, which are located in this interval. These experiments provide direct physiological and causal evidence that p27 has tumor suppressive functions independent of its role regulating cell proliferation.     

Setting of the candidate exposure conditions in an individualized tumor response testing (ITRT) toward an individual chemotherapy for esophageal cancer

Background  To apply the collagen gel droplet embedded culture-drug sensitivity test (CD-DST) technique to individualized tumor response testing (ITRT) in esophageal cancer, optimal exposure conditions that would permit us to predict the clinical response to the low-dose cisplatin (CDDP)/5-fluorouracil (5-FU) regimen and docetaxel (DOC) regimen were determined. Methods  CD-DST was performed on 35 surgical specimens and 27 endoscopic biopsy samples from patients with esophageal squamous cell cancer. The reported blood AUC values were reproduced, or an in vitro concentration-response curve was constructed, for each regimen to determine exposure conditions using surgical specimens. For endoscopic samples, we estimated the evaluation rate (of CD-DST) and compared in vitro sensitivity with clinical response in measurable lesions to validate the exposure conditions determined. Results  Overall, data were evaluated in 75.8% of the cases (94.3% in surgical specimens and 51.9% in endoscopic biopsy samples). Modeled exposure conditions were 5-FU 2.0 μg/ml/120 h with a cutoff inhibition rate (IR) of 50% for the low-dose CDDP/5-FU regimen and DOC 0.04 μg/ml/120 h with a cutoff IR of 50% for the DOC regimen. Conclusions  Although the study failed to compare in vitro results with clinical outcomes in measurable lesions, and the practical usefulness and applicability of the technique remain to be demonstrated, CD-DST might be a useful tool in selecting optimal chemotherapeutic agents.     

cAMP-dependent protein kinase regulates in ovo camp level of the Xenopus oocyte: Evidence for an intracellular feedback mechanism

Microinjection of cAMP-dependent protein kinase inhibitor (1.8 μM) increases the cAMP level of Xenopus oocyte. Its effect was observed in full-grown (stage VI) as well as in vitellogenic (stage IV) oocytes. In contrast the inhibitor I₁ of protein phosphatase-1 blocks cAMP accumulation. Progesterone (1 μM) decreases the cAMP level in control and in PKI-treated oocytes of both stages. These results show that cAMP concentration is regulated by a cAMP-dependent phosphorylation indicating the presence of a feedback mechanism. The feedback control is disrupted when oocyte is induced to mature by progesterone.     

In situ growth of a PEG-like polymer from the C?terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation

This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (R_h): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (R_h: 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles.     

Questioning the preclinical paradigm: natural,extreme biology as an alternative discovery platform: A New Modest Proposal for Preventing the Children of the World from Being Burdened,Like Their Parents,by Disease and Making Beneficial Therapies (shamelessly co-opted from Dr. Swift)

The pace at which science continues to advance is astonishing. From cosmology, microprocessors, structural engineering, and DNA sequencing our lives are continually affected by science-based technology. However, progress in treating human ailments, especially age-related conditions such as cancer and Alzheimer''s disease, moves at a relative snail''s pace. Given that the amount of investment is not disproportionately low, one has to question why our hopes for the development of efficacious drugs for such grievous illnesses have been frustratingly unrealized. Here we discuss one aspect of drug development –rodent models – and propose an alternative approach to discovery research rooted in evolutionary experimentation. Our goal is to accelerate the conversation around how we can move towards more translative preclinical work.