Euthanasia. Figures for "slow euthanasia" should be included in data on physician assisted suicide.
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Double Effect Principle↗
Biomedical subjects
M H Irwin
Publications and source records attributed to M H Irwin.
Isolation and microinjection of somatic cell-derived mitochondria and germline heteroplasmy in transmitochondrial mice.
At present, there are no means for creation of relevant animal models of human mitochondrial DNA (mtDNA)-based diseases in a directed fashion. As an initial step towards this end, we have developed a microinjection technique for transfer of isolated, viable mitochondria between two mouse species. Previously, we reported detection, by nested PCR with species-specific primer sets, of Mus spretus mtDNA in Mus musculus domesticus blastocyts following zygote microinjection and culture. We now report the production of transmitochondrial founder mice, and germline transmission of the heteroplasmic state in a maternal lineage. Heteroplasmic mice produced by this technique will be useful in the study of mitochondrial dynamics and may hasten the creation of animal models of human mtDNA-based diseases.
Animals↗
In vitro fertilization in mice: Strain differences in response to superovulation protocols and effect of cumulus cell removal.
Strain differences have proven to be crucial components in mouse in vitro fertilization (IVF) and superovulatory protocols. To maximize the yield of IVF-derived mouse eggs, a series of experiments was conducted using different injection timing intervals for administration of pregnant mare serum gonadotropin (PMSG) and hCG to induce follicular development and ovulation. Strains were chosen that were representative of those commonly used in genetic engineering experimentation. These strains included ICR outbred, C57BL/6 inbred, and B6SJLF1 hybrid (C57BL/6J x SJL/J F1) mice. Females were superovulated using 4 PMSG/hCG/IVF timing regimens (group), with sperm obtained from males of the same strain. Group designations were based on the following PMSG/hCG and hCG/oocyte collection intervals, respectively: Group 1, 55 and 21.5 h; Group 2, 60 and 14.5 h; Group 3, 55 and 14.5 h; Group 4, 48 and 14.5 h. After overnight culture of ova, fertilization rates (development to the 2-cell stage) were assessed. A logistic regression was performed using indicator variables for both strain and group. There was a significant strain influence on ova fertilization rate, based on the coefficients of mouse strain (ICR, beta = -1.1067, P = 8E-17 and C57BL/6, beta = -0.5172, P = 8E-06). Additionally, group affected the proportion of fertilized ova obtained (coefficient of Group 1, beta = -1.3152, P = 0.00 and Group 3, beta = 0.9531, P = 3E-12). From the coefficients for the interaction terms, the effect of groups varies across mouse strain. Therefore, the treatment that produces the highest fertilization rate is related to and contingent upon the strain of mouse. In the second study, the Group 3 protocol was used to evaluate fertilization differences between cumulus-intact and cumulus-free oocytes. Again, there was a significant strain influence on ova fertilization rate based on the coefficients of mouse strain (ICR, beta = -2.6639, P = 0.00; C57BL/6, beta = -2.5114, P = 0.00). However, there was no difference between Cumulus and No Cumulus groups (cumulus coefficient, beta = 0.1640, P = 0.59872), indicating that there was no affect of cumulus presence on fertilization rate. In summary, responses to standardized mouse IVF protocols vary significantly. The efficiency of IVF procedures can be optimized between and within specific mouse strains by the timing of superovulatory regimens. However, absence of cumulus cells during the IVF procedure does not adversely affect fertilization rate.
Journal Article↗
Mitochondria transfer into mouse ova by microinjection.
A method for mitochondria isolation and interspecific transfer of mitochondria was developed in mice. Mitochondria were isolated from Mus spretus liver samples for microinjection into fertilized ova obtained from superovulated M. musculus domesticus females. Electron microscopic observations of mitochondria preparations used for microinjection demonstrated intact mitochondrial vesicles with little microsomal contamination. Species-specific nested PCR primers complementary to sequence differences in the mitochondrial DNA D-loop region revealed high rates of successful transfer of foreign mitochondria after isolation and injection into zygotes cultured through the blastocyst stage of embryonic development. Of 217 zygotes, 67 survived mitochondria injection and 23 out of 37 zygotes developed were at the blastocyst-stage of embryonic development after 4.5 days of in vitro culture. All 23 of these blastocysts contained detectable levels of foreign mitochondria. These results represent an initial step in developing a model system to study mitochondrial dynamics and development of therapeutic strategies for human metabolic diseases affected by aberrations in mitochondrial function or mutation.
Animals↗
Astrocyte growth, reactivity, and the target of the antiproliferative antibody, TAPA.
Reactive astrocytes form a scar after injury to the CNS that many investigators believe contributes to the lack of functional regeneration. In the present study, we identify an astrocytic membrane protein that appears to play an important role in reactive gliosis and scar formation. Cultures of rat astrocytes were used as a model system to produce and to screen monoclonal antibodies that would alter cell growth. One antibody, AMP1, was identified that depresses the mitotic activity of cultured glial cells and alters their morphology. Expression cloning reveals that the antigen on the external surface of the cultured glial cells has a high degree of homology with the human lymphocyte protein called Target of the Anti-Proliferative Antibody (TAPA-1; this rat protein will be referred to as rTAPA). rTAPA is a member of the tetramembrane-spanning superfamily of proteins and, as with other members of this family of proteins, rTAPA is associated with the regulation of cellular interactions and mitotic activity. After an injury to the cerebral cortex, there is a dramatic increase in AMP1 immunoreactivity that is spatially restricted to the reactive astrocytes at the glial scar. This change represents an upregulation of a membrane protein, rTAPA, that is approximately equal to the increase observed for glial fibrillary acidic protein. The high levels of rTAPA at the site of CNS injury and the AMP1 antibody perturbation studies indicate that rTAPA may play a prominent role in the response of astrocytes to injury and in glial scar formation.
Actinin↗
Identification of transgenic mice by PCR analysis of saliva.
As an alternative to surgically obtaining samples (e.g., tail or tissue biopsy, toe dock, or blood sampling) from weanling mice to screen for transgene integration or other genetic monitoring procedures, we offer a simpler, nonsurgical method. A small amount of saliva, obtained from weanling mice by oral wash using a plastic pipet tip, contains enough oral epithelial cells and lymphocytes to yield sufficient DNA for nested primer polymerase chain reaction (PCR) analysis. The procedure can be repeated many times with minimal stress to the animal, in contrast to tissue biopsy procedures such as tail cutting. Sample analysis is rapid and straightforward; saliva is applied to sample collection paper and then purified using a solid phase DNA purification system. The paper, containing purified DNA, is added directly to PCR cocktail for the first round of amplification. For weanling mice, in the second round of amplification, a small amount of product from the first round is removed and added to PCR cocktail containing the second set of primers. With adult mice, an adequate volume of saliva may be obtained (dependent upon the sensitivity of the particular reaction) to eliminate the need for second-round amplification with nested primers. This technique is reliable, does not require organic solvents, and is more humane than protocols currently in use. Furthermore, this technique could replace hundreds of thousands of surgical biopsies on rodents annually, which are performed for both transgene determination and genetic monitoring procedures.
Animals↗
The psychological impact of sexual abuse of Native American boarding-school children.
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Adolescent↗
Distribution and characteristics of a 90 kDa protein, KG-CAM, in the rat CNS.
The distribution of a 90 kDa protein, termed KG-CAM, was examined in the developing and adult rat central nervous system (CNS) using the monoclonal antibody 11-59. The amino acid sequence of this protein revealed a sequence homology with a group of chick cell adhesion molecules from the immunoglobulin superfamily: DM-GRASP; SC1; and BEN. Immunolabeling of cells cultured from the embryonic and neonatal rat brain demonstrates that the protein recognized by 11-59 is on the external surface of a subpopulation of neurons and a limited population of glial cells. When the 11-59 antibody was used to stain sections of the adult brain and spinal cord, a number of different structures were labeled. The most intense immunoreactivity was found in the somatosensory system, the basal ganglia, the cortex, the olfactory system, and the circumventricular organs. One of the more interesting aspects of KG-CAM is the spatially and temporally regulated patterns of expression observed during the development of the CNS. For example, the dendrites of layer II pyramidal cells in the granular retrosplenial cortex are immunopositive for 11-59 while the dendrites are in the process of bundling in layer I, but not before bundling begins or after the process is completed. These findings reveal the varied roles of this adhesion molecule in the developing brain and spinal cord, as well as its potential role in the maintenance of the structural integrity of the adult CNS.
Activated-Leukocyte Cell Adhesion Molecule↗
The upregulation of a glial cell surface antigen at the astrocytic scar in the rat.
The upregulation of a 106-kDa glial protein was examined using indirect immunohistochemical methods. The protein is recognized by the monoclonal antibody AMP1. Previous studies demonstrated that the AMP1 antigen is found on the external surface of cultured astrocytes and is involved in stabilizing adhesive interactions between these cells. In sections of injured adult rat brain, the spatial distribution of the AMP1 antigen correlated with the region of reactive gliosis. The relative intensity of immunofluorescence indicates that the AMP1 antigen is dramatically upregulated at the gliotic scar. The correlation between AMP1 antigen upregulation and reactive gliosis suggests that this molecule is critical to the process of CNS scar formation.
Animals↗
Laminin cleavage by activated human neutrophils yields proteolytic fragments with selective migratory properties.
We studied the interactions between human neutrophils, as well as the purified human neutrophil serine proteases elastase (HNE) and cathepsin G (HNCG), and laminin. Our results show that intact laminin and two proteolytic fragments generated by HNE bind to neutrophils and stimulate cell migration. Domain-specific antilaminin monoclonal antibodies, rotary shadowing electron microscopy, and Western blotting mapped the two promigratory fragments on the laminin cross to the apical three-armed region and long arm, respectively. In contrast, a fragment derived from the terminal ends of short arms neither bound to neutrophils nor stimulated migration. When neutrophils embedded in a reconstituted basement membrane gel were activated with phorbol myristate acetate, several stable, proteolytic laminin fragments were released into supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that these fragments appeared identical to those generated after digestion of soluble laminin with HNE and HNCG. Furthermore, release of laminin fragments by embedded neutrophils was inhibited by diisopropyl fluorophosphate, and duplicated by incubating the basement membrane gel with purified HNE and HNCG. Our findings therefore suggest that neutrophils, through release of HNE and HNCG, are capable of digesting basement membrane laminin in vivo. In addition, the release of laminin fragments from damaged basement membranes may promote neutrophil migration and thereby accelerate inflammatory processes.
Adult↗
A novel cell adhesion molecule, G-CAM, found on cultured rat glia.
Using a monoclonal antibody (mAb), designated AMP1, a novel cell adhesion molecule was identified on rat astrocytes and oligodendrocytes. When confluent monolayers of cultured rat astrocytes were labeled with AMP1, the antigen was discretely localized to the cell surface in regions of cell-cell contacts. The antibody did not label embryonic rat cortical neurons plated on monolayers of neonatal astrocytes, indicating that the antigen is neither present on cultured neurons nor does it reorganize on the glial surface under the neurons. On immunoblots of astrocytic or brain proteins, mAb AMP1 recognized a 106 kDa protein. In the present paper, data are presented demonstrating that the AMP1 antigen is a cell adhesion molecule and is distinct from all the known cell adhesion molecules present on astrocytes: N-CAM, N-cadherin, or members of the beta 1 integrin family. We have tentatively termed this molecule 'glial cell adhesion molecule' (G-CAM).
Animals↗
The structure and macromolecular organization of type IX collagen in cartilage.
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Amino Acid Sequence↗
Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G.
To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manner yielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss of laminin epitopes from basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation.
Cathepsin G↗
Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains.
To examine the ultrastructural distribution of laminin within kidney basement membranes, we prepared rat anti-mouse laminin mAbs to use in immunolocalization experiments. Epitope domains for these mAbs were established by immunoprecipitation, immunoblotting, affinity chromatography, and rotary shadow EM. One mAb bound to the laminin A and B chains on blots and was located to a site approximately 15 nm from the long arm-terminal globular domain as shown by rotary shadowing. Conjugates of this long arm-specific mAb were coupled to horseradish peroxidase (HRP) and intravenously injected into mice. Kidney cortices were fixed for microscopy 3 h after injection. HRP reaction product was localized irregularly within the renal glomerular basement membrane (GBM) and throughout mesangial matrices. In addition, this mAb bound in linear patterns specifically to the laminae rarae of basement membranes of Bowman's capsule and proximal tubule. This indicates the presence of the long arm immediately beneath epithelial cells in these sites. The laminae densae of these basement membranes were negative by this protocol. In contrast, the lamina rara and densa of distal tubular basement membranes (TBM) were both heavily labeled with this mAb. A different ultrastructural binding pattern was seen with eight other mAbs, including two that mapped to different sites on the short arms by rotary shadowing and five that blotted to a large pepsin-resistant laminin fragment (P1). These latter mAbs bound weakly or not at all to GBM but all bound throughout mesangial matrices. In contrast, discrete spots of HRP reaction product were seen across all layers of Bowman's capsule BM and proximal TBM. These same mAbs, however, bound densely across the full width of distal TBM. Our findings therefore show that separate strata of different basement membranes are variably immunoreactive to these laminin mAbs. The molecular orientation or integration of laminin into the three dimensional BM meshwork therefore varies with location. Alternatively, there may be a family of distinct laminin-like molecules distributed within basement membranes.
Animals↗
Monoclonal antibodies to type X collagen. Biosynthetic studies using an antibody to the amino-terminal domain.
Monoclonal antibodies to chick type X collagen have been used to study the structure, biosynthesis, and location of type X in cartilage. The antibodies were produced by injecting purified type X collagen into female SJL/J mice and then fusing their spleen cells with Sp2/0 myeloma cells. Hybridoma culture supernatants were screened for antibodies to type X collagen by enzyme-linked immunosorbent assay and Western blots. Positive supernatants did not cross-react with other collagen types (I, II, IX, XI) or with fibronectin. Three monoclonal antibodies were chosen for further characterization. Two of them (1A6 and 6F6) recognize a pepsin-sensitive domain of type X collagen. Rotary shadowing showed that 1A6 and 6F6 both recognize the same end of type X, probably the aminoterminal non-triple helical domain. Amino acid sequencing of the intact protein and of the epitope-containing peptide confirmed that the antibody recognition sites for 1A6 and 6F6 are within the amino-terminal domain. Monoclonal antibody 2B3 reacts with the pepsinized (45 kDa) and weakly with the nonpepsinized (59 kDa) forms of type X collagen. The monoclonal antibodies were used for immunolocalization of type X in hypertrophic chondrocytes and reacted only with tissue samples from areas undergoing endochondral ossification, e.g. growth plate and fracture callus. Antibody 6F6, when coupled to Sepharose, selectively binds to type X collagen from cell and organ cultures. In a pulse-chase experiment, no processing of the 59-kDa form of type X could be detected. Two components with molecular masses of approximately 70 and 85 kDa, arising from a disulfide-bonded aggregate, were synthesized by both the permanent and calcifying cartilage organ cultures but did not react with the antibody, suggesting that these proteins are not related to type X. In summary, the pulse-chase results and the immune precipitation with monoclonal antibody 6F6 did not detect biosynthetic precursors larger than 59 kDa or proteolytically processed forms of type X.
Amino Acid Sequence↗
Future of IPPNW. International Physicians for the Prevention of Nuclear War.
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International Cooperation↗