gebhart/patrepeval huggingface dataset:
Characterization and enrichment of fetal rat hepatoblasts by immunoadsorption (“panning”) and fluorescence-activated cell sorting (1994)

We developed methods for enriching fetal hepatoblasts by combining panning and multiparametric fluorescence-activated cell sorting. In unpurified, dissociated fetal liver cell suspensions of embryonic age day 15, 3.2% ± 1.3% and 2.5% ± 0.7% cells expressed albumin and α-fetoprotein, respectively. The remainder exhibited a hemopoietic, endothelial or stromal cell phenotype. Cells were panned first with an antibody to red blood cells to remove erythroid cells and then with monoclonal antibodies OX-43/OX-44 to remove hemopoietic and endothelial cells. This procedure eliminated 84% of fetal hepatic cells, with enrichment of the remainder for albumin or α-fetoprotein expression (up to sixfold increase). Flow cytometric analysis of unlabeled cells revealed two populations, which differed in granularity and autofluorescence. After panning, fluorescence-activated cell sorting for agranular cells yielded OX-43/44-positive cells that were essentially all hemopoietic precursor cells or OX-43/44-negative cells that were mostly hemopoietic precursor cells, along with 3.0% ± 0.7% α-fetoprotein-positive cells. In contrast, sorting for granular, OX-43/44-negative cells enriched for predominantly α-fetoprotein-positive, parenchymal precursor cells (75.1% ± 4.7%). Multiparametric flow cytometric analysis of the expression of an oval cell antigen, OC.3, which is a bile duct and putative liver stem cell marker, showed that most OC.3-positive cells coexpressed OX-43/OX-44 and morphologically were hemopoietic precursor cells. However, approximately 30% of the OX-43/44-negative, granular cells expressed OC.3. Although the physiological significance of OC.3-positive hepatoblasts remains to be determined, the ability to isolate distinct liver cell populations by means of fluorescence-activated cell sorting should facilitate further studies. In addition, because panning alone produced significantly enriched populations of fetal hepatoblasts, applications not requiring further cell purification could be performed with this simple technique. (Hepatology 1994;19:999-1006.)
Human articular surface chondrocytes initiate alkaline phosphatase and type X collagen synthesis in suspension culture (1992)

The type X collagen is a short chain collagen associated with calcific cartilage and/or the expression of the hypertrophic chondrocyte phenotype. In articular cartilage, type X collagen is restricted to the basal zone of calcified cartilage adjacent to the subchondral bone. However, during pathological change such as in osteoarthritis, the synthesis of type X collagen becomes more widespread but never extends to the articular surface. Using immunocytochemistry and fluorography of newly synthesised collagens, we report that surface articular chondrocytes (which occupy the uppermost 10-15% of the tissue depth) from normal human cartilage initiate de novo synthesis of both type X collagen and alkaline phosphatase when maintained in suspension culture.
Pathway-Based Identification of Biomarkers for Targeted Therapeutics: Personalized Oncology with PI3K Pathway Inhibitors (2010)

Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K-mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway-relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40(Thr246). Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)-deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40(Thr246) positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKT(Thr308), the phospho-PRAS40(Thr246) epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug-specific biomarkers toward development of patient-tailored treatments.
A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate. (1985)

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), encoded by the aroA locus, is a target site of glyphosate inhibition in bacteria. A glyphosate-resistant aroA allele has been cloned in Escherichia coli from a mutagenized strain of Salmonella typhimurium. Subcloning of this mutant aroA allele shows the gene to reside on a 1.3-kilobase segment of S. typhimurium DNA. Nucleotide sequence analysis of this mutant gene indicates a protein-coding region 427 amino acids in length. Comparison of the mutant and wild type aroA gene sequences reveals a single base pair change resulting in a Pro to Ser amino acid substitution at the 101st codon of the protein. A hybrid gene fusion between mutant and wild type aroA gene sequences was constructed. 5-Enolpyruvylshikimate-3-phosphate synthase was prepared from E. coli cells harboring this construct. The glyphosate-resistant phenotype is shown to be associated with the single amino acid substitution described above.
Nutritionally Induced Diabetes in Desert Rodents as Models of Type 2 Diabetes: Acomys cahirinus (Spiny Mice) and Psammomys obesus (Desert Gerbil) (2006)

The dietary effects of hyperglycemia increasingly result in type 2 diabetes in humans. Two species, the spiny mice (Acomys cahirinus) and the desert gerbil (Psammomys obesus), which have different metabolic responses to such effects, are discussed. Spiny mice exemplify a pathway that leads to diabetes without marked insulin resistance due to low supply of insulin on abundant nutrition, possibly characteristic of a desert animal. They respond with obesity and glucose intolerance, beta-cell hyperplasia, and hypertrophy on a standard rodent diet supplemented with fat-rich seeds. The accompanying hyperglycemia and hyperinsulinemia are mild and intermittent but after a few months, the enlarged pancreatic islets suddenly collapse, resulting in loss of insulin and ketosis. Glucose and other secretagogues produce only limited insulin release in vivo and in vitro, pointing to the inherent disability of the beta-cells to respond with proper insulin secretion despite their ample insulin content. On a 50% sucrose diet there is marked lipogenesis with hyperlipidemia without obesity or diabetes, although beta-cell hypertrophy is evident. P.obesus is characterized by muscle insulin resistance and the inability of insulin to activate the insulin signaling on a high-energy (HE) diet. Insulin resistance imposes a vicious cycle of Hyperglycemia and compensatory hyperinsulinemia, leading to beta-cell failure and increased secretion of proinsulin. Ultrastructural studies reveal gradual disappearance of beta-cell glucokinase, GLUT 2 transporter, and insulin, followed by apoptosis of beta-cells. Studies using the non-insulin-resistant HE diet-fed animals maintained as a control group are discussed. The insulin resistance that is evident to date in the normoglycemic state on a low-energy diet indicates sparing of glucose fuel in muscles of a desert-adapted animal for the benefit of glucose obligatory tissues. Also discussed are the effect of Psammomys age on the disabetogenicity of the HE diet; the impaired function of several components of the insulin signal transduction pathway in muscles, which reduces the availability of GLUT4 transporter; the testing of several antidiabetic modalities for the prevention of nutritional diabetes in Psammomys; and various complications related to the diabetic condition.
Molecular Cloning and Nucleotide Sequence of the Gramicidin S Synthetase 1 Gene1 (1989)

The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.
Immunodominance analysis of CTL responses to influenza PR8 virus reveals two new dominant and subdominant Kb-restricted epitopes (1996)

In the present study, a systematic analysis of the influenza (Flu) PR8 determinants recognized by H-2b mice was undertaken. A single Db-restricted immunodominant epitope (NP(366)) was previously known in this system. Twenty-three different Flu PR8-derived peptides that bound either Kb or Db molecules in vitro were identified. Sixteen were immunogenic following peptide immunization of C57BL/6 mice, yet CTL induced by peptide immunization recognized PR8-infected target cells only in the case of the NP(366) and NS2(114) epitopes. Similarly, CTL responses following whole-PR8 virus immunization were detected only for the same two determinants. CTL recognizing these dominant epitopes had high avidity for peptide-pulsed target cells, with 5 to 200 pM of peptide required for 30% specific lysis. In contrast, most (80%) of the remaining epitopes were recognized with lower avidity (30% effective concentration in the range of 0.4-50 nM). Repeated in vitro stimulation of primary CTL cultures revealed one additional Kb-restricted epitope (M1(128)). This peptide bound Kb with high affinity (4.6 nM) and induced CTL that effectively recognized PR8-infected cells. These results suggest that 1) this epitope is produced by natural processing in relatively high amounts and 2) low precursor frequency might be related to the subdominant status of the M1(128) epitope. Taken together, these results illustrate the crucial contributions of MHC-binding capacity, and T cell repertoire availability, to the shaping of the repertoire of CTL specificities for Flu Ag virus.
Immune Responses to AAV-Vectors, the Glybera Example from Bench to Bedside (2014)

Alipogene tiparvovec (Glybera(®)) is an adeno-associated virus serotype 1 (AAV1)-based gene therapy that has been developed for the treatment of patients with lipoprotein lipase (LPL) deficiency. Alipogene tiparvovec contains the human LPL naturally occurring gene variant LPL(S447X) in a non-replicating viral vector based on AAV1. Such virus-derived vectors administered to humans elicit immune responses against the viral capsid protein and immune responses, especially cellular, mounted against the protein expressed from the administered gene have been linked to attenuated transgene expression and loss of efficacy. Therefore, a potential concern about the use of AAV-based vectors for gene therapy is that they may induce humoral and cellular immune responses in the recipient that may impact on efficacy and safety. In this paper, we review the current understanding of immune responses against AAV-based vectors and their impact on clinical efficacy and safety. In particular, the immunogenicity findings from the clinical development of alipogene tiparvovec up to licensing in Europe will be discussed demonstrating that systemic and local immune responses induced by intra-muscular injection of alipogene tiparvovec have no deleterious effects on clinical efficacy and safety. These findings show that muscle-directed AAV-based gene therapy remains a promising approach for the treatment of human diseases.
Simultaneous Quantitation of Delta-9-tetrahydrocannabinol (THC) and 11-Nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in Serum by GC/MS Using Deuterated Internal Standards and Its Application to a Smoking Study and Forensic Cases (1992)

A new procedure for the simultaneous detection of delta-9-tetrahydrocannabinol (THC) and its major metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in serum has been evaluated. The method combines rapid, efficient, solid-phase extraction and simple derivatization by methylation. Analysis and quantitation is performed by gas chromatography/mass spectrometry (GC/MS) using deuterated cannabinoids as internal standards (IS). Reproducibility and sensitivity of the method are good. The procedure is applied to serum specimens collected from a smoking study with 24 volunteers and 212 forensic cases. Results are interpreted based upon the current knowledge about THC metabolism and pharmacokinetics.
Major histocompatibility complex class I allele‐specific peptide libraries: Identification of peptides that mimic an H‐Y T cell epitope (1994)

We describe a novel method for screening large libraries of random peptides for T cell antigens. Two libraries were constructed, containing fixed amino acids representing the major histocompatibility complex (MHC) class I anchor residues for H-2Kb-restricted octamers and H-2Db-restricted nonamers. Peptides from the Kb-restricted library (KbL: SXIXFXXL) and the Db-restricted library (DbL: XXXXNXXXIM) specifically stabilize empty Kb and Db molecules, respectively. The libraries contain peptides that mimic several H-2b-restricted cytotoxic T lymphocyte epitopes, and 21 mimotopes for a Db-restricted H-Y epitope were isolated. A degenerate synthetic peptide of limited complexity containing the identified H-Y sequence motif was found to be similar to the natural H-Y epitope by reverse-phase high performance liquid chromatography analysis. This peptide is also capable of immunizing female mice against male splenocytes. Several applications for MHC-restricted peptide libraries are discussed.
A short-pulsed laser cleaning system for EUVL tool (2007)

A traditional method of reticle protection, using a pellicle, is thought to be difficult to apply to EUVL tool. There is a possibility that some particles adhere to the surface of a reticle. In order to resolve this reticle issue, we are investigating in-situ short-pulsed laser cleaning system. In order to confirm whether it can be applied to EUVL tool or not, we have to verify experimentally that it is possible to remove particles in vacuum without any damage to a Mo/Si multilayer. In this work, as a short-pulsed laser, a Q-switched YAG Laser (wavelength: 266~1064 nm, pulse duration time: ~7 ns) has been used. As experimental results, at the pressure on the order of 10<sup>-3</sup> Pa, around 100% removal rate to PSL (organic) particles can be achieved without particular damage using a DUV laser. And also, it is found that a laser cleaning method in vacuum is easier to remove particles on a surface than that in atmosphere and for SiO<sub>2</sub> and Ni (inorganic) particles, this method is particularly effective. This reason can be explained that the drag force exerted to a particle by the surrounding gas molecules is neglected at the pressure on the order of 10<sup>-3</sup> Pa because a gas is in free molecule regime.
Amplification of a Cloned Chinese Hamster Dihydrofolate Reductase Gene After Transfer into a Dihydrofolate Reductase-Deficient Cell Line (1983)

We have transformed a dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary cell line to the DHFR+ phenotype with a recombinant cosmid (cH1) containing a functional Chinese hamster DHFR gene (J.D. Milbrandt et al., Mol. Cell. Biol. 3:1266-1273, 1983). After exposure of cells to successive increases in methotrexate, we have isolated a resistant cell line (JSH-1) that grows in 1 microM methotrexate. We show here that JSH-1 contains 300 to 500 copies of the integrated cosmid and that these copies are located predominantly at one position on a chromosome identified as Z5a. Hybridization analysis of restriction digests of genomic DNA indicates that the cosmid has been integrated intact into the genome and that upon amplification, the original cosmid/genomic junction fragments are also amplified in JSH-1. Furthermore, the pattern of amplified bands observed in ethidium bromide-stained gels indicates that the unit amplified sequence (amplicon) may be as large as 120 to 135 kilobases and therefore includes considerable amounts of flanking DNA in addition to the 45 kilobases of integrated cosmid. We also show that the protein overproduced by the amplified cosmid in JSH-1 comigrates with the 21,000-dalton polypeptide characteristic of the methotrexate-resistant cell line (CHOC 400) from which cH1 was cloned. However, the DHFR mRNA species overproduced in JSH-1 appear to be larger than those detected in CHOC 400, indicating that not all of the normal transcription and processing signals are preserved in the integrated recombinant cosmid.
A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5 (2000)

HIV-1 entry into CD4(+) cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.
Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences. (1997)

Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015, respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of .005 (*1x2), .013 (*2x2), and .001 (*4x2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T1957C), *2B (additional C2558T), and *4E (additional C2938T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EM/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment.
The Immunopathology of Psoriasis (1996)

Psoriasis affects approximately 2% of the population in Western countries. Although clinically rather monomorphic, the disease presents with a number of phenotypically distinct and heterogeneous subgroups showing differences in their pathogenetic pathways. Patients with type I (early onset) psoriasis demonstrate inflammatory lesions with epidermal hyperproliferation and the presence of activated T cells as well as intraepidermal polymorphs as principal features. In contrast to pustular types of psoriasis, these patients show genetic susceptibility and strong association with MHC class I and class-I haplotypes. There is evidence for a T-cell-mediated pathomechanism leading to a large spectrum of regulatory mediators including cytokines and growth factors as well as lipid mediators which are abnormally expressed. Pathogenetically psoriasis shows features in common with chronic relapsing T-cell-mediated diseases including Crohn's disease, rheumatoid arthritis and others.
Amyloid-β oligomers: their production, toxicity and therapeutic inhibition (2002)

Despite extensive genetic and animal modelling data that support a central role for the amyloid beta-protein (A beta) in the genesis of Alzheimer's disease, the specific form(s) of A beta which causes injury to neurons in vivo has not been identified. In the present study, we examine the importance of soluble, pre-fibrillar assemblies of A beta as mediators of neurotoxicity. Specifically, we review the role of cell-derived SDS-stable oligomers, their blocking of hippocampal long-term potentiation in vivo and the finding that this blocking can be prevented by prior treatment of oligomer-producing cells with gamma-secretase inhibitors.
Small Dense LDL Cholesterol and Coronary Heart Disease: Results from the Framingham Offspring Study (2010)

Despite 4-fold greater cholesterol-lowering therapy use, CHD patients had mean LDL-C concentrations above the LDL-C goal of <2.6 mmol/L (<100 mg/dL). Although women with CHD had higher sdLDL-C concentrations than controls, this difference was not seen in men. These findings may explain some of the high residual risk of future CHD events in CHD patients.