gebhart/patrepeval huggingface dataset:
How to make bispecific antibodies. (2000)

This protocol describes the production of bispecific F(ab')2 antibody derivatives (BsAbs) by the linking of two Fab' fragments via their hinge region SH groups using the bifunctional crosslinker o-phenylenedimaleimide (o-PDM) as described by Glennie et al. (1,2). The procedure is illustrated in Fig. 1. The first step is to obtain F(ab')2 from the two parent IgG antibodies. Methods for digestion of IgG to F(ab')2 are described in Chapter 25. Fab' fragments are then prepared from the two F(ab')(2) species by reduction with thiol, thus exposing free SH groups at the hinge region (three SH-groups for mouse IgG1 and IgG2a antibodies) (see Note 1 ). One of the Fab' species (Fab'-A) is selected for alkylation with o-PDM. Because o-PDM has a strong tendency to crosslink adjacent intramolecular SH-groups, two of the three hinge SH-groups will probably be linked together, leaving a single reactive maleimide group available for conjugation (Fig. 1; see Note 2 ). Excess o-PDM is then removed by column chromatography, and the Fab'-A(mal) is mixed with the second reduced Fab' (Fab'-B) under conditions favoring the crosslinking of the maleimide and SH groups. When equal amounts of the two parent Fab' species are used, the major product is bispecific F(ab')(2), resulting from the reaction of one Fab'-A(mal) with one of the SH groups at the hinge of Fab'-B. Increasing the proportion of Fab'-A(mal) in the reaction mixture results in a significant amount of F(ab')(3) product by the reaction of two molecules of Fab'-A(mal) with two free SH-groups at the hinge of a single Fab'-B molecule (see Note 3). The remaining free SH groups on Fab'-B are alkylated, and the F(ab')(2) bispecific antibody product (Fab'-A x Fab'-B) is separated by gel filtration chromatography. Each stage of the procedure is checked by HPLC.
Site-Directed Mutations of Human Growth Hormone that Selectively Modify Its Lactogenic Activity and Binding Properties (1990)

Two novel analogs of human (h) GH, 1) Des-7-hGH (Arg8Met, Asp11Ala) in which the Arg8 was substituted by Met and Asp11 by Ala, and 2) bovine (b) GH/hGH hybrid II (MetAla 1-13/14-191, Ala11Asp) composed of 13 N-terminal amino acid of bGH and elongated by two amino acids (Met-Ala-1-13) and 14-191 amino acids of hGH, were constructed and expressed in Escherichia coli. CD spectra indicated that the alpha-helix content of the purified proteins was similar to that of the native hormone. Both analogs retained their full ability to stimulate the proliferation of Nb2 lymphoma cells, and their binding to the lactogen receptors in homogenate of Nb2 cells and in microsomal fraction from bovine lactating mammary gland was only slightly reduced. However, their ability to bind to the somatogen receptors in intact IM-9 lymphocytes and bovine liver was reduced by 7- to 11-fold (bGH/hGH hybrid II) and 20- to 30-fold (Des-7-hGH). Both analogs were able to down-regulate the respective lactogen and somatogen receptors in intact Nb2 and IM-9 cells. The galactopoietic activity of both analogs in the lactating bovine mammary explants bioassay was almost completely abolished, and the bGH/hGH hybrid II exhibited a remarkable antagonistic activity. These results further indicate that the lactogen receptors in different species or organs are not identical. We have shown that the new recombinant analogs of hGH that recognize both somatogen and lactogen receptors but have modified postreceptor effects are helpful in elucidating these differences.
Dendritic Cells Injected Via Different Routes Induce Immunity in Cancer Patients (2001)

Dendritic cells (DC) represent potent APCs that are capable of generating tumor-specific immunity. We performed a pilot clinical trial using Ag-pulsed DC as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly injections of DC enriched and activated from their PBMC. DC were cocultured ex vivo with recombinant mouse prostatic acid phosphatase as the target neoantigen. Following enrichment, DC developed an activated phenotype with up-regulation of CD80, CD86, and CD83 expression. During culture, the DC maintained their levels of various adhesion molecules, including CD44, LFA-1, cutaneous lymphocyte-associated Ag, and CD49d, up-regulated CCR7, but lost CD62 ligand and CCR5. In the absence of CD62 ligand, such cells would not be expected to prime T cells efficiently if administered i.v. due to their inability to access lymphoid tissue via high endothelial venules. To assess this possibility, three patient cohorts were immunized with Ag-pulsed DC by i.v., intradermal (i.d.), or intralymphatic (i.l.) injection. All patients developed Ag-specific T cell immune responses following immunization, regardless of route. Induction of IFN-gamma production, however, was seen only with i.d. and i.l. routes of administration, and no IL-4 responses were seen regardless of route, consistent with the induction of Th1-type immunity. Five of nine patients who were immunized by the i.v. route developed Ag-specific Abs compared with one of six for i.d. and two of six for i.l. routes. These results suggest that while activated DC can prime T cell immunity regardless of route, the quality of this response and induction of Ag-specific Abs may be affected by the route of administration.
Protein Ladder Sequencing (1993)

A new approach to protein sequencing is described. It consists of two steps: (i) ladder-generating chemistry, the controlled generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted laser-desorption mass spectrometry. Each amino acid was identified from the mass difference between successive peaks, and the position in the data set defined the sequence of the original peptide chain. This method was used to directly locate a phosphoserine residue in a phosphopeptide. The protein ladder sequencing method lends itself to very high sample throughput at very low per cycle cost.
Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate (1996)

The polyhydroxybutyrate (PHB) synthase gene of the bacterium Alcaligenes eutrophus was used to construct a yeast plasmid which enabled expression of the functional synthase enzyme in Saccharomyces cerevisiae. Cells transformed with the synthase plasmid accumulated up to 0.5% of cell dry weight as PHB, with accumulation occurring in the stationary phase of batch growth. The identity of PHB in recombinant yeast cells was confirmed with 1H-NMR spectra of chloroform-extracted cell material. In addition, freeze-fracture electron microscopy revealed cytoplasmic granules exhibiting plastic deformations characteristic for PHB. GC results indicated a low background level of PHB in the wild-type strain, but intact polymer could not be detected by 1H-NMR. Formation of PHB in the recombinant strain implies the participation of native yeast enzymes in the synthesis of D-3-hydroxybutyryl-CoA (3-HB-CoA). Inhibition studies with cerulenin indicated that the fatty acid synthesis pathway is not involved in PHB precursor formation. Wild-type cell-free extracts showed D-3-HB-CoA dehydrogenase activity [150-200 nmol min-1 (mg protein)-1] and acetoacetyl-CoA thiolase activity [10-20 nmol min-1 (mg protein)-1], which together could synthesize monomer from acetyl-CoA. PHB accumulation was simultaneous with ethanol production, suggesting that PHB can act as an alternate electron sink in fermentative metabolism. We propose that PHB synthesis in recombinant yeast is catalysed by native cytoplasmic acetoacetyl-CoA thiolase, a native beta-oxidation protein possessing D-3-HB-CoA dehydrogenase activity and heterologous PHB synthase.
Phosphorylcholine-Coated Metallic Stents in Rabbit Iliac and Porcine Coronary Arteries (1998)

The arterial wall reaction to phosphorylcholine-coated metal stents was examined in rabbits and pigs. Compared to non-coated stents, no significant difference was found by angiography and histology. We conclude that although phosphorylcholine-coating does not provoke arterial neointima formation or decrease luminal diameter compared to stainless steel stents, the coating does not seem to reduce restenosis.
A matched filter bound analysis of single- and multi-carrier DS CDMA communications (2002)

The equivalence and differences between the single wideband DS CDMA and multicarrier DS CDMA (MC-CDMA) transmissions are shown in this paper through a matched filter bound analysis. It is shown that by combining the three matched filter outputs in the three band MC-CDMA case, the output statistic is the same as output for each bit in the single wideband case for an arbitrary delay profile. Even though their performances are equivalent in most practical cases, it is shown that single wideband CDMA can more effectively use time diversity from interleaving especially when infinite interleaving can be achieved.
Application of high pressure deuterium annealing for improving the hot carrier reliability of CMOS transistors (2000)

We present the effect of high pressure deuterium annealing on hot carrier reliability improvements of CMOS transistors. High pressure annealing increases the rate of deuterium incorporation at the SiO/sub 2//Si interface. We have achieved a significant lifetime improvement (90/spl times/) from fully processed wafers (four metal layers) with nitride sidewall spacers and SiON cap layers. The improvement was determined by comparing to wafers that were annealed in a conventional hydrogen forming gas anneal. The annealing time to achieve the same level of improvement is also significantly reduced. The increased incorporation of D at high pressure was confirmed by the secondary ion mass spectrometry characterization.
Titanium Dioxide Nanoparticles Induce DNA Damage and Genetic Instability In vivo in Mice (2009)

Titanium dioxide (TiO(2)) nanoparticles are manufactured worldwide in large quantities for use in a wide range of applications including pigment and cosmetic manufacturing. Although TiO(2) is chemically inert, TiO(2) nanoparticles can cause negative health effects, such as respiratory tract cancer in rats. However, the mechanisms involved in TiO(2)-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. The present study investigates TiO(2) nanoparticles-induced genotoxicity, oxidative DNA damage, and inflammation in a mice model. We treated wild-type mice with TiO(2) nanoparticles in drinking water and determined the extent of DNA damage using the comet assay, the micronuclei assay, and the gamma-H2AX immunostaining assay and by measuring 8-hydroxy-2'-deoxyguanosine levels and, as a genetic instability endpoint, DNA deletions. We also determined mRNA levels of inflammatory cytokines in the peripheral blood. Our results show that TiO(2) nanoparticles induced 8-hydroxy-2'-deoxyguanosine, gamma-H2AX foci, micronuclei, and DNA deletions. The formation of gamma-H2AX foci, indicative of DNA double-strand breaks, was the most sensitive parameter. Inflammation was also present as characterized by a moderate inflammatory response. Together, these results describe the first comprehensive study of TiO(2) nanoparticles-induced genotoxicity in vivo in mice possibly caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the growing use of TiO(2) nanoparticles, these findings raise concern about potential health hazards associated with TiO(2) nanoparticles exposure.
Direct non-cell autonomous Pax6 activity regulates eye development in the zebrafish (2007)

Abstract Background Modifications in Pax6 homeogene expression produce strong eye phenotypes. This suggested to us that eye development might be an appropriate model to verify if homeoprotein intercellular passage has important functions in early development. Similar to other homeoproteins, Pax6 has two domains that enable secretion and internalization by live cells and, thus, intercellular passage. In principle, a straightforward way to test the hypothesis would be to mutate one of the two sequences to produce a 'cell autonomous only' Pax6. However, this was not possible because these sequences are in the homeodomain and their modification would affect Pax6 transcriptional properties. We have thus developed an approach aimed at blocking Pax6 only in the extracellular milieu of developing zebrafish embryos. Results A first strategy was to inject a one-cell embryo with a mRNA encoding a secreted single-chain anti-Pax6 antibody. A second, complementary, strategy was to inject a Pax6 antibody in the blastula extracellular milieu. In both cases, 'dissymmetric eyes', 'one eye only' and 'no eye' phenotypes were produced. In most cases, lens phenotypes paralleled retina malformations. Although eye phenotypes were analyzed 30 hours post-fertilization, there was a strong correlation between early eye field asymmetry, early asymmetry in Pax6 expression and later-occurring eye malformations. Several controls were introduced, demonstrating that the effect is specific to Pax6 and cannot be explained by intracellular antibody activities. Conclusion This study supports the hypothesis that the Pax6 transcription factor is also a signaling molecule with direct non-cell autonomous activity.
Upregulation of the Voltage-Gated Sodium Channel β2 Subunit in Neuropathic Pain Models: Characterization of Expression in Injured and Non-Injured Primary Sensory Neurons (2005)

The development of abnormal primary sensory neuron excitability and neuropathic pain symptoms after peripheral nerve injury is associated with altered expression of voltage-gated sodium channels (VGSCs) and a modification of sodium currents. To investigate whether the beta2 subunit of VGSCs participates in the generation of neuropathic pain, we used the spared nerve injury (SNI) model in rats to examine beta2 subunit expression in selectively injured (tibial and common peroneal nerves) and uninjured (sural nerve) afferents. Three days after SNI, immunohistochemistry and Western blot analysis reveal an increase in the beta2 subunit in both the cell body and peripheral axons of injured neurons. The increase persists for >4 weeks, although beta2 subunit mRNA measured by real-time reverse transcription-PCR and in situ hybridization remains unchanged. Although injured neurons show the most marked upregulation,beta2 subunit expression is also increased in neighboring non-injured neurons and a similar pattern of changes appears in the spinal nerve ligation model of neuropathic pain. That increased beta2 subunit expression in sensory neurons after nerve injury is functionally significant, as demonstrated by our finding that the development of mechanical allodynia-like behavior in the SNI model is attenuated in beta2 subunit null mutant mice. Through its role in regulating the density of mature VGSC complexes in the plasma membrane and modulating channel gating, the beta2 subunit may play a key role in the development of ectopic activity in injured and non-injured sensory afferents and, thereby, neuropathic pain.
Dysfunction of CFTR bearing the delta F508 mutation. (1993)

The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in patients with cystic fibrosis (CF). The most common CF-associated mutation is deletion of phenylalanine at residue 508, CFTR delta F508. When expressed in heterologous cells, CFTR bearing the delta F508 mutation fails to progress through the normal biosynthetic pathway and fails to traffic to the plasma membrane. As a result, CFTR delta F508 is mislocalized and is not present in the apical membrane of primary cultures of airway epithelia. Consequently, the apical membrane of CF airway epithelia is Cl- -impermeable, a defect that probably contributes to the pathogenesis of the disease.
VASO-DILATORS IN HIGH BLOOD PRESSURE (1909)

Journal Article VASO-DILATORS IN HIGH BLOOD PRESSURE Get access EDWIN MATTHEW EDWIN MATTHEW Search for other works by this author on: Oxford Academic PubMed Google Scholar QJM: An International Journal of Medicine, Volume os2, Issue 3, April 1909, Pages 261–278, https://doi.org/10.1093/oxfordjournals.qjmed.a069217 Published: 01 April 1909
Mechanical Model of Vertical Nanowire Cell Penetration (2013)

Direct access into cells' interiors is essential for biomolecular delivery, gene transfection, and electrical recordings yet is challenging due to the cell membrane barrier. Recently, molecular delivery using vertical nanowires (NWs) has been demonstrated for introducing biomolecules into a large number of cells in parallel. However, the microscopic understanding of how and when the nanowires penetrate cell membranes is still lacking, and the degree to which actual membrane penetration occurs is controversial. Here we present results from a mechanical continuum model of elastic cell membrane penetration through two mechanisms, namely through "impaling" as cells land onto a bed of nanowires, and through "adhesion-mediated" penetration, which occurs as cells spread on the substrate and generate adhesion force. Our results reveal that penetration is much more effective through the adhesion mechanism, with NW geometry and cell stiffness being critically important. Stiffer cells have higher penetration efficiency, but are more sensitive to NW geometry. These results provide a guide to designing nanowires for applications in cell membrane penetration.
In Vitro and In Vivo Characterization of Helicobacter hepaticus Cytolethal Distending Toxin Mutants (2004)

Helicobacter hepaticus expresses a member of the cytolethal distending toxin (CDT) family of bacterial cytotoxins. To investigate the role of CDT in the pathogenesis of H. hepaticus, transposon mutagenesis was used to generate a series of isogenic mutants in and around the cdtABC gene cluster. An H. hepaticus transposon mutant with a disrupted cdtABC coding region no longer produced CDT activity. Conversely, a transposon insertion outside of the cluster did not affect the CDT activity. An examination of these mutants demonstrated that CDT represents the previously described granulating cytotoxin in H. hepaticus. Challenge of C57BL/6 interleukin 10(-/-) mice with isogenic H. hepaticus mutants revealed that CDT expression is not required for colonization of the murine gut. However, a CDT-negative H. hepaticus mutant had a significantly diminished capacity to induce lesions in this murine model of inflammatory bowel disease.
Significance of the Melanocortin 1 and Endothelin B Receptors in Melanocyte Homeostasis and Prevention of Sun-Induced Genotoxicity (2016)

The membrane bound melanocortin 1 receptor (MC1R), and the endothelin B receptor (ENDBR) are two G-protein coupled receptors that play important roles in constitutive regulation of melanocytes and their response to ultraviolet radiation (UVR), the main etiological factor for melanoma. The human MC1R is a Gs protein-coupled receptor, which is activated by its agonists α-melanocyte stimulating hormone (α-melanocortin; α-MSH) and adrenocorticotropic hormone (ACTH). The ENDBR is a Gq coupled-receptor, which is activated by Endothelin (ET)-3 during embryonic development, and ET-1 postnatally. Pigmentation and the DNA repair capacity are two major factors that determine the risk for melanoma. Activation of the MC1R by its agonists stimulates the synthesis of eumelanin, the dark brown photoprotective pigment. In vitro studies showed that α-MSH and ET-1 interact synergistically in the presence of basic fibroblast growth factor to stimulate human melanocyte proliferation and melanogenesis, and to inhibit UVR-induced apoptosis. An important function of the MC1R is reduction of oxidative stress and activation of DNA repair pathways. The human MC1R is highly polymorphic, and MC1R variants, particularly those that cause loss of function of the expressed receptor, are associated with increased melanoma risk independently of pigmentation. These variants compromise the DNA repair and antioxidant capacities of human melanocytes. Recently, activation of ENDBR by ET-1 was reported to reduce the induction and enhance the repair of UVR-induced DNA photoproducts. We conclude that α-MSH and ET-1 and their cognate receptors MC1R and ENDBR reduce the risk for melanoma by maintaining genomic stability of melanocytes via modulating the DNA damage response to solar UVR. Elucidating the response of melanocytes to UVR should improve our understanding of the process of melanomagenesis, and lead to effective melanoma chemoprevention, as well as therapeutic strategies.
A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida. (1985)

After the acrosome reaction, the PH-20 surface antigen of guinea pig sperm migrates from its original location on the posterior head surface to a new location on the inner acrosomal membrane (Myles, D.G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). We have isolated three monoclonal antibodies (MAbs) of the IgG1 subclass, PH-20, PH-21, and PH-22, that bind to the PH-20 antigen. The PH-20 MAb strongly inhibited (approximately 90%) sperm binding to the guinea pig egg zona pellucida at saturating antibody concentrations (greater than 20 micrograms/ml). Half-maximal inhibition of sperm binding to the zona was obtained with approximately 2 micrograms/ml PH-20 MAb. The PH-21 MAb at saturating concentration (50 micrograms/ml) partially inhibited (approximately 45%) sperm-zona binding, and the PH-22 MAb (50 micrograms/ml) did not inhibit (0%) sperm-zona binding. Essentially the same amounts of the three MAbs were bound to sperm under the conditions where inhibition (PH-20, PH-21) or no inhibition (PH-22) of sperm-zona binding was observed, which indicates that the different levels of inhibition did not arise from different levels of MAb binding. Competition binding assays with 125I-labeled MAbs showed that PH-21 binding to sperm was not affected by the binding of PH-20 or PH-22. However, that PH-20 and PH-22 blocked each other's binding to sperm suggests that their recognized determinants may be relatively close to one another. The results indicate that the migrating PH-20 antigen has a required function in sperm binding to the zona pellucida and that the PH-20 MAb affects is active site.
Immunotherapy with autologous tumor cells engineered to secrete Tag7/PGRP, an innate immunity recognition molecule (2004)

We can conclude that secretion of the Tag7 protein by genetically modified cells can induce mobilization of antigen-presenting cells and innate effectors. Memory mechanisms are mediated by T cell response. For the first time our results demonstrate that local secretion of Tag7-the molecule involved in innate immunity-may play an important role in the induction of effective antitumor response in mice.
`Eph'ective signaling: forward, reverse and crosstalk (2003)

The Eph receptors comprise the largest group of receptor tyrosine kinases and are found in a wide variety of cell types in developing and mature tissues. Their ligands are the ephrins, a family of membrane-bound proteins found in lipid rafts. In the past decade, Eph receptors and ephrins have been implicated in a vast array of cellular processes. Unlike other receptor tyrosine kinases, however, the Eph receptors seem to be geared towards regulating cell shape and movement rather than proliferation. Studies have uncovered intricate signaling networks that center around the ligand-receptor complex, and this may account for the broad repertoire of functions of Eph proteins. Deciphering the bi-directional pathways emanating from an Eph receptor-ephrin complex will not only help us to understand basic biological processes, but may also provide important insight into disease.