Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose.

Xenogeneic immunizations have the advantage of detecting a wide range of antigenic determinants because many commonly occurring proteins have diverged significantly during the course of evolution and are thus antigenic in other species. The broadness of xenogeneic responses, however, means that the antisera they produce are usually complex and require extensive absorptions to make them specific for a single antigen. This problem has now been overcome by generating hybridomas producing monoclonal antibodies (Kohler & Milstein 1975). These permit dissection ofthe xenogeneic response so that large amounts of individual antibodies can be obtained, each of which recognizes only one of the determinants recognized by a broadly reactive conventional antiserum. Williams et al. (1977) used hybridoma monoclonal antibodies obtained after immunizations of mice with rat cells to study rat cell-surface antigens present on subpopulations of rat lymphocytes, i.e., differentiation antigens. Springer et al. (1978a) and Stern et al. (1978) used a similar approach to study mouse lymphocyte antigens. They prepared monoclonal antibodies by immunizing rats with mouse lymphocytes and showed that these monoclonals recognized previously undetected mouse cell surface determinants including a glycoprotein antigen that appears to be specific for macrophages (Springer et al. 1978b). Trowbridge (1978) also used rat anti-mouse immunizations to generate a monoclonal antibody against the non-polymorphic lymphocyte surface antigen T200.

Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens.

This review is focused on purinergic neurotransmission, i.e., ATP released from nerves as a transmitter or cotransmitter to act as an extracellular signaling molecule on both pre- and postjunctional membranes at neuroeffector junctions and synapses, as well as acting as a trophic factor during development and regeneration. Emphasis is placed on the physiology and pathophysiology of ATP, but extracellular roles of its breakdown product, adenosine, are also considered because of their intimate interactions. The early history of the involvement of ATP in autonomic and skeletal neuromuscular transmission and in activities in the central nervous system and ganglia is reviewed. Brief background information is given about the identification of receptor subtypes for purines and pyrimidines and about ATP storage, release, and ectoenzymatic breakdown. Evidence that ATP is a cotransmitter in most, if not all, peripheral and central neurons is presented, as well as full accounts of neurotransmission and neuromodulation in autonomic and sensory ganglia and in the brain and spinal cord. There is coverage of neuron-glia interactions and of purinergic neuroeffector transmission to nonmuscular cells. To establish the primitive and widespread nature of purinergic neurotransmission, both the ontogeny and phylogeny of purinergic signaling are considered. Finally, the pathophysiology of purinergic neurotransmission in both peripheral and central nervous systems is reviewed, and speculations are made about future developments.

/pdf/physiology-and-pathophysiology-of-purinergic-1z94g2hz0f.pdf

Physiology and Pathophysiology of Purinergic Neurotransmission

Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins.

There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors deorphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review.

https://pharmrev.aspetjournals.org/content/pharmrev/58/3/281.full.pdf

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

Monoclonal Antibodies: Principles and Practice

Use of staphylococcal protein A as an immunological reagent

Advances in somatic cell hybridization techniques have made it possible to generate hybrid cell lines producing monospecific antibodies directed at desired antigenic determinants (1). In this paper a modification of the cell fusion procedure (2,3) was used to recover stable hybrid cell lines secreting IgG antibodies to: (a) mouse major histocompatibility complex (MHC) alloantigens (H-2K and I-A); and (b) mouse immunoglobulin (Ig) allotypes (Ig-1b, Ig-5a, and Ig-5b).

Properties of Monoclonal Antibodies to Mouse Ig Allotypes, H-2, and Ia Antigens

Osteoblasts mineralize bone matrix by promoting hydroxyapatite crystal formation and growth in the interior of membrane-limited matrix vesicles (MVs) and by propagating the crystals onto the collagenous extracellular matrix. Two osteoblast proteins, tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) are involved in this process. Mutations in the TNAP gene result in the inborn error of metabolism known as hypophosphatasia, characterized by poorly mineralized bones, spontaneous fractures, and elevated extracellular concentrations of inorganic pyrophosphate (PP(i)). PP(i) suppresses the formation and growth of hydroxyapatite crystals. PP(i) is produced by the nucleoside triphosphate pyrophosphohydrolase activity of a family of isozymes, with PC-1 being the only member present in MVs. Mice with spontaneous mutations in the PC-1 gene have hypermineralization abnormalities that include osteoarthritis and ossification of the posterior longitudinal ligament of the spine. Here, we show the respective correction of bone mineralization abnormalities in knockout mice null for both the TNAP (Akp2) and PC-1 (Enpp1) genes. Each allele of Akp2 and Enpp1 has a measurable influence on mineralization status in vivo. Ex vivo experiments using cultured double-knockout osteoblasts and their MVs demonstrate normalization of PP(i) content and mineral deposition. Our data provide evidence that TNAP and PC-1 are key regulators of the extracellular PP(i) concentrations required for controlled bone mineralization. Our results suggest that inhibiting PC-1 function may be a viable therapeutic strategy for hypophosphatasia. Conversely, interfering with TNAP activity may correct pathological hyperossification because of PP(i) insufficiency.

https://www.pnas.org/content/pnas/99/14/9445.full.pdf

James W. Goding

Papers

Monoclonal Antibodies: Principles and Practice

Use of staphylococcal protein A as an immunological reagent

Properties of Monoclonal Antibodies to Mouse Ig Allotypes, H-2, and Ia Antigens

Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization

Conjugation of antibodies with fluorochromes: modifications to the standard methods.