Consensus agreement regarding protocol issues discussed during the mouse lymphoma workshop: Portland, Oregon, May 7, 1994.
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Animals↗
Biomedical subjects
J Clements
Publications and source records attributed to J Clements.
Is it possible to assess the impact of abuse on children with pervasive developmental disorders?
Although it is now recognized that children may exhibit widespread symptoms of stress following exposure to abuse or neglect, the impact of traumatic experiences on children with pervasive developmental disorders has received little attention. The present paper describes a strategy devised to assess the long-term effects of abuse on a group of children who had attended a specialist autistic school where physical and emotional mistreatment of pupils was well documented. Because most of the children had very limited communication skills, the evidence relied heavily on retrospective parental reports. Despite the problems inherent in using such data it was possible to derive reliable measures that could be used to test hypotheses about predicted patterns of behavioral change, based on work with other children who have been abused. In most cases, too, parental reports could be corroborated by information from other sources. Consistent patterns in both the nature and timing of behavioral disturbances were found, which seemed to relate specifically to the period of abuse at the school.
Activities of Daily Living↗
Gene mutation assays in mammalian cells.
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Animals↗
Integrin alpha 4 beta 1-mediated melanoma cell adhesion and migration on vascular cell adhesion molecule-1 (VCAM-1) and the alternatively spliced IIICS region of fibronectin.
The integrin receptor alpha 4 beta 1 (also known as VLA-4) binds two different ligands, the endothelial cell surface protein vascular cell adhesion molecule-1 (VCAM-1) and the extracellular matrix component fibronectin. Three distinct sites in fibronectin are recognized by alpha 4 beta 1. Two of these (represented by peptides CS1 and CS5) are present in the alternatively spliced IIICS region and lie in separate, independently spliced segments of this region. A third site resides in the adjacent constitutively expressed HepII domain. Recombinant proteins containing the HepII domain and different splice variants of the IIICS have been generated and compared for their ability to mediate cell attachment, spreading and migration. The activity of these proteins has also been compared with that of a recombinant soluble form of VCAM-1 (rsVCAM-1). All the recombinant proteins supported A375-SM human melanoma cell attachment and spreading in an alpha 4 beta 1-dependent manner, but had varied adhesive activities with rsVCAM-1 > fibronectin variants containing the CS1 sequence >> other fibronectin variants. Low concentrations of rsVCAM-1 and CS1-containing fibronectin variants effectively supported cell migration in a trans-filter assay; however, cell motility was retarded at high concentrations of the same proteins. Fibronectin variants lacking CS1 supported little or no migration. To obtain further insight into the molecular basis of this varied adhesive activity, apparent dissociation constants for each of the recombinant proteins were measured using a solid phase receptor-ligand binding assay. The results revealed a hierarchy of ligand affinities that mirrored their adhesive activity (rsVCAM-1 > fibronectin variants containing CS1 >> other fibronectin variants).
Alternative Splicing↗
Binding of a soluble alpha beta T-cell receptor to superantigen/major histocompatibility complex ligands.
The genes for the alpha and beta chains of a murine T-cell receptor were truncated just prior to the portions encoding the transmembrane regions and introduced into baculovirus by recombination. Insect cells infected with the virus secreted a soluble form of the receptor that could be purified to homogeneity. This soluble receptor reacted with a set of six monoclonal antibodies originally raised to different epitopes on the natural transmembrane-region-containing receptor and bound with appropriate specificity to a cell surface complex of the human major histocompatibility complex class II molecule DR1 with the bacterial superantigen staphylococcal enterotoxin B.
Amino Acid Sequence↗
Processing of tumour necrosis factor-alpha precursor by metalloproteinases.
Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory and immunomodulatory cytokine implicated in inflammatory conditions such as rheumatoid arthritis, Crohn's disease, multiple sclerosis and the cachexia associated with cancer or human immunodeficiency virus infection. TNF-alpha is initially expressed as a 233-amino-acid membrane-anchored precursor which is proteolytically processed to yield the mature, 157-amino-acid cytokine. The processing enzyme(s) which cleave TNF-alpha are unknown. Here we show that the release of mature TNF-alpha from leukocytes cultured in vitro is specifically prevented by synthetic hydroxamic acid-based metalloproteinase inhibitors, which also prevent the release of TNF-alpha into the circulation of endotoxin challenged rats. A recombinant, truncated TNF-alpha precursor is cleaved to biologically active, mature TNF-alpha by several matrix metalloproteinase enzymes. These results indicate that processing of the TNF-alpha precursor is dependent on at least one matrix metalloproteinase-like enzyme, inhibition of which represents a novel therapeutic mechanism for interfering with TNF-alpha production.
Animals↗
Production of soluble MHC class II proteins with covalently bound single peptides.
The alpha beta T-cell receptors (TCRs) react with complex ligands composed of peptides bound to major histocompatibility complex (MHC) proteins. In the absence of foreign antigens the peptides bound to MHC molecules come from the proteins of the host itself. Interactions between TCRs and these self-peptide-MHC ligands work positively to drive T-cell development in the thymus and negatively to delete or inactivate T cells with potential self-reactivity. On the cell surface, MHC proteins are associated with many different self peptides, making it impossible to know which self peptide was involved in positive or negative interactions with a particular T cell. These studies as well as in vitro studies on TCR-peptide-MHC interactions would be aided by a means of producing MHC molecules containing a single peptide. We have tackled this problem for MHC class II proteins by genetically attaching the peptide by a flexible peptide linker to the amino terminus of the class II beta-chain. Here we report that a secreted, soluble form of this covalent peptide-MHC complex can be expressed in insect cells. The peptide is engaged by the peptide-binding groove of the secreted MHC molecule and this complex is recognized by T cells bearing receptors specific for that combination.
Amino Acid Sequence↗
Competitive binding of vascular cell adhesion molecule-1 and the HepII/IIICS domain of fibronectin to the integrin alpha 4 beta 1.
The integrin receptor alpha 4 beta 1 binds to two different ligands, the extracellular matrix glycoprotein fibronectin and the endothelial cell surface protein vascular cell adhesion molecule-1 (VCAM-1). Using probes derived from each ligand and a variety of cell adhesion and ligand-receptor binding assays, we have investigated the relationship between the mechanisms of fibronectin and VCAM-1 interaction with alpha 4 beta 1. CS1 peptide, which represents the dominant active site from the HepII/IIICS recognition domain in fibronectin, was found to inhibit VCAM-1-dependent adhesion in three different assays: MOLT-4 T lymphoblastic leukaemia cell attachment to immobilized recombinant soluble VCAM-1 (rsVCAM-1), MOLT-4 cell attachment to monolayers of VCAM-1-transfected COS-1 cells, and A375-SM melanoma cell spreading on immobilized rs VCAM-1. Half-maximal inhibition required CS1 concentrations of 1.7-3.0 mg/ml, some 3-7-fold higher than that needed to autoinhibit adhesion to CS1-IgG conjugate. Using a more sensitive solid-phase receptor-ligand binding assay, CS1 was found to be a potent inhibitor of the binding of rsVCAM-1 to alpha 4 beta 1 (half-maximal inhibition at 13 micrograms/ml). In agreement with cell-based assays, severalfold lower concentrations of CS1 were required to inhibit binding of recombinant HepII/IIICS region of fibronectin (half-maximal inhibition at 3 micrograms/ml). VCAM-1-alpha 4 beta 1 binding was blocked not only by CS1 peptide but also by the recombinant HepII/IIICS region of fibronectin. Kinetic analysis of CS1 inhibition of VCAM-1 binding revealed that it was directly competitive in nature, indicating that VCAM-1 and fibronectin recognize either identical or spatially overlapping binding sites on alpha 4 beta 1. The implications of these results for the future design of VCAM-1 antagonists are discussed.
Amino Acid Sequence↗
Parallels in T lymphocyte development between lpr and normal mice.
The unusual phenotype and function of lpr T cell subsets has considerably aided the determination of various stages in normal T lymphocyte development through the identification of parallel populations. This has included the description of memory T cells that express high levels of CD44. Lpr mature (CD4+ and CD8+) T cells are all CD44hi and produce large amounts of several cytokines, in contrast to normal murine peripheral T cells. However, the minor CD44hi subset of normal memory T cells produces similarly high levels of cytokines. The expression of intermediate density surface T cell antigen receptor (TCR)-alpha beta by lpr CD4-, CD8- (CD4-8-) cells guided the identification of a minor subset of Heat Stable Antigen--normal CD4-8- thymocytes that also expresses TCR-alpha beta. The unresponsiveness of lpr CD4-8- T cells correlates closely with their absence of CD2 expression. This is paralleled in normal mice by CD2- and CD2+ subsets of thymocytes and gut lymphocytes in which the proliferative capacity also segregates with CD2 expression. Finally, lpr CD4-8- T cells bear many similarities to anergic T cells, including high expression of p59fyn, lack of IL-2 production, and recovery of function following induction of cell cycling in the presence of IL-2. The developmental block in lpr CD4-8- T cells has therefore provided considerable insight into normal T cell development and function.
Animals↗
Distinct binding specificities of integrins alpha 4 beta 7 (LPAM-1), alpha 4 beta 1 (VLA-4), and alpha IEL beta 7.
Integrin receptors are important for regulating lymphocyte recirculation and recruitment to sites of inflammation. Transfectants of the B cell lymphoma 38C13 were generated that differ exclusively in the expression of integrin beta 1 or beta 7 subunits allowing for a functional comparison of lymphocyte Peyer's patch HEV adhesion molecule 1 (LPAM-1) (alpha 4 beta 7) and very late antigen 4 (VLA-4) (alpha 4 beta 1) in an identical cellular environment. Whereas 38-beta 7 transfectants bound to purified and cellular mucosal addressin cell adhesion molecule (MAdCAM-1), unstimulated 38-beta 1 cells failed to bind MAdCAM-1. Treatment of 38-beta 1 cells with Mn2+ but not with PMA induced low level binding to MAdCAM-1. MAdCAM-1 adhesion of 38-beta 7 cells was constitutive and not enhanced by Mn2+ treatment. Similarly, MAdCAM-1-dependent adhesion to mucosal high endothelial venules was shown for 38-beta 7 but not for 38-beta 1 cells. The results therefore establish the LPAM-1-MAdCAM-1 interaction as the functionally dominant adhesion pathway for regulating lymphocyte homing to mucosal sites. Nonetheless, the activated VLA-4 on some lymphocytes may be involved in MAdCAM-1 recognition or promote binding to MAdCAM-1 in other tissues. By contrast, 38-beta 7 and 38-beta 1 transfectants did not differ in their binding capacity for vascular cell adhesion molecule 1 (VCAM-1) or fibronectin and LPAM-1 did not display any preference for interacting with either MAdCAM-1 or VCAM-1. LPAM-1 may therefore contribute significantly to cellular functions previously attributed to VLA-4. Interestingly, functional analysis of the intraepithelial lymphocyte integrin alpha IEL beta 7 which is structurally related to LPAM-1 did not reveal detectable binding activity for MAdCAM-1, VCAM-1, or fibronectin.
Animals↗
Glandular kallikreins and prostate-specific antigen are expressed in the human endometrium.
Glandular or tissue kallikrein and prostate-specific antigen (PSA) are members of the human kallikrein (KLK) multigene family of enzymes. Various components of the glandular kallikrein-kinin system (kallikrein, low molecular weight kininogen, bradykinin) have been recently shown to be present or active in the human endometrium. We have used the reverse transcriptase-polymerase chain reaction (RT-PCR) with universal KLK primers to demonstrate kallikrein gene expression in this tissue. On Southern blot analysis with gene-specific oligonucleotide probes, we have detected expression of the three human KLK genes--KLK1 (kallikrein), KLK2 (or hGK1) and KLK3 (PSA). The expression of KLK1 and KLK3 was further confirmed by sequence analysis of three different endometrial PCR products. These findings confirm the presence of a local kallikrein-kinin system in the human endometrium. The significance of the novel expression of KLK2 and KLK3 (PSA), previously thought to be prostate-specific genes, in the endometrium is unclear. This family of enzymes must now be considered potential local regulators of uterine function.
Base Sequence↗
Glandular kallikrein gene expression in the human uterus.
1. Kallikrein enzyme activity has been previously reported in the uterus of several species and implicated in implantation and parturition. In order to provide further evidence for a local kallikrein-kinin system in this tissue, we wished to determine if the gene encoding kallikrein (KLK1) was expressed in the human uterus and determine the pattern of its expression across the menstrual cycle. 2. Reverse-transcriptase polymerase chain reaction (RT-PCR) of endometrial and myometrial total RNA coupled with Southern blot analysis showed that KLK1 was expressed in the human endometrium and myometrium. Endometrial expression of KLK1 was confirmed by DNA sequence analysis of the PCR products. Kallikrein was also localized by immunocytochemistry, primarily in the glandular epithelium of the endometrium. 3. Quantitative RT-PCR of 37 endometrial samples ranging from day 1 to 29 from across the menstrual cycle showed significantly higher KLK1 (kallikrein) expression from the mid proliferative to the early secretory phase compared with the late secretory and menstrual phases. 4. We have demonstrated for the first time that KLK1, the gene encoding glandular kallikrein, is expressed in the human uterus. The increase in endometrial KLK1 gene expression during the proliferative phase of the menstrual cycle suggests a role for kallikrein in the preparation of the endometrial lining for implantation.
Blotting, Southern↗
Safety evaluation of esperase.
Esperase is a proteolytic enzyme preparation that can be used as a processing aid in the food industry. The following studies were performed to establish safety for the consumer: oral toxicity study (13 wk) in the rat; teratogenicity study in the rat; gene mutation assays in Salmonella typhimurium and mammalian cells in vitro, and chromosome aberration assay in vitro. General toxicity was low; the effects seen were attributed to proteolytic activity and the loading with sodium chloride. Neither of these factors will be relevant to consumers of the processed food. There was no evidence of effects on pregnancy outcome or mutagenic potential. When these results are considered together with existing knowledge of the production organism and the chemical and microbiological characterization of the enzyme preparation, they indicate that Esperase will be safe for its intended application in food processing.
Abnormalities, Drug-Induced↗
The need for continuing education.
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Attitude of Health Personnel↗
The genotoxicity of the anti-cancer drug mitoxantrone in somatic and germ cells of Drosophila melanogaster.
The novel antineoplastic drug mitoxantrone was studied for its genotoxic effects in Drosophila melanogaster. In male germ cells, the clinical preparation Novantrone, the dihydrochloride salt of mitoxantrone, did not induce sex-linked recessive lethal mutations in feeding and injection experiments with adult flies, although statistically the results were inconclusive rather than truly negative. However, the free base mitoxantrone was weakly, but significantly genotoxic in this test (0.14% lethals/mM exposure concentration); this is most probably the result of prolonged exposure. On the other hand, both forms of mitoxantrone assayed were clearly genotoxic in the somatic mutation and recombination test of the wing. This test assays the cells of the proliferating imaginal wing discs of larvae. Depending on the feeding method used, the overall clone induction frequency was in the range of about 2-6 x 10(-5) per cell and cell generation and per mM exposure dose. Correction of these frequencies according to mean clone size led to slightly higher estimates (by about 5-25% higher). Although the majority of the clone induction events are due to mitotic recombination, a significant proportion can be attributed to mutational events (gene and chromosome mutations). The genotoxicity of mitoxantrone seems to depend mainly on impaired DNA synthesis in cycling cells owing to the compound's ability to inhibit topoisomerase II by intercalation into DNA.
Animals↗
Mutations defining functional regions of the superantigen staphylococcal enterotoxin B.
Staphylococcal enterotoxin B (SEB) is both a superantigen and toxin. As a superantigen, SEB can bind to major histocompatibility complex (MHC) class II molecules to form a ligand for alpha/beta T cell receptors bearing particular V beta elements. As a toxin, SEB causes rapid weight loss in mice sometimes leading to death. We show here that both of these functions map to the NH2-terminal portion of the toxin. Three regions were identified: one important in MHC class II binding, one in T cell recognition, and one in both functions. These results support the conclusion that the toxicity of SEB is related to massive T cell stimulation and release of cytokine mediators and show that the residues interacting with MHC and the T cell receptor are intertwined.
Amino Acid Sequence↗
A re-evaluation of the tissue-specific pattern of expression of the rat kallikrein gene family.
We have used reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and insitu hybridization to further define the tissue and cell-specific pattern of expression of the rat kallikrein gene family. rKlk1 gene expression has been unequivocally demonstrated in the rat heart, ovary and testis. Similarly, rKlk3, 4 and 9 gene expression has been demonstrated in the testis/ovary, heart and testis respectively. Insitu hybridization has identified the expression of kallikrein gene family members to specific cell-types in the rat ovary (granulosa cell) and testis (germ cell).
Animals↗
Prolonged transgene expression in rodent lung cells.
We tested the efficiency of several different cationic liposome formulations, complexed to one of two different chloramphenicol acetyltransferase (CAT) reporter plasmids, in transfecting freshly isolated, highly purified rat lung alveolar type II cells, alveolar macrophages, and three different human lung carcinoma cell lines, as well as NIH 3T3 cells, a rapidly dividing, transformed mouse fibroblast line. Our results demonstrated that several different cationic liposome formulations can mediate high-level CAT gene expression in all the cell types tested. Electron microscopic analysis confirmed that cationic liposome-DNA complexes are avidly bound and internalized by lung cells. The time course of expression of transfected genes in nontransformed cell types with low mitotic indices, such as type II cells, is poorly characterized. NIH 3T3 cells expressed maximal CAT activity by day 4 following transfection, with virtual disappearance of activity by day 11. Conversely, type II cells expressed maximal CAT activity between days 5 and 11, and CAT activity was still clearly present 35 days after transfection. Southern blot analysis of DNA isolated from transfected type II cells revealed that the CAT gene was largely present in an extrachromosomal form, rather than integrated into genomic DNA. These observations indicate that following cationic liposome-mediated transfection, rat alveolar type II cells (the majority of which do not divide in culture) can express transfected genes for prolonged periods, apparently mediated by expression of the transgene in an episomal form.
3T3 Cells↗