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Influence of thymosin and the supernatant of cultured mouse thymus reticuloepithelial cell on mouse immune responses.

Effects of the humoral factor extracted from the calf thymus (thymosin: Goldstein et al.) and the supernatant of cultured mouse thymus reticuloepithelial cell (TRC) on immunological functions of the mouse lymphocytes were studied, and the following conclusions were drawn: Thymosin treatment did not alter the capacity to form anti-SRBC antibody in adult mice which were lethally irradiated and reconsituted with bone marrow cells. When the spleen cells from JCL-ICR mice thymectomized within 24 hr after birth were cultured with thymosin or supernatant of cultured ICR-TRC, the Thy 1 positive cells were increased. A significant increase in the mitogenic (PHA, Con A) reactivity of BALB/C spleen cells was observed when these cells were preincubated in medium containing thymosin for 24 hr. The NZB spleen cells, however, exhibited spleen cells showed a slightly potentiated response only to Con A. Similarly, thymosin enhanced the MLC reaction of the spleen cells from BALB/C mice with preincubation for 2 hr, but not enhanced that from NZB mice.

Animals↗

The mouse vertebrae: changes in the morphology of mouse vertebrae exhibit specific patterns over limited numbers of vertebral levels.

The mouse vertebrae from the cervix to the tip of the tail were characterized and anatomical features that have been lacking were added to the classical description. The vertebrae consist of six long-range and fourteen short-range substructures, with the foveal process being a newly identified substructure. The caudal transverse process, cranial hemal process and hemal ridge are substructures that are clearly defined in the mouse. Each long-range and short-range substructure has several specific morphological features such as length, width, area, shape and angle. These features exibit a crescendo, plateau or decrescendo pattern over a limited number of vertebral segments that ranges from just a few to twenty. The variety of substructural combinations and the constant changes in the morphological features lead to the fact that no single vertebra has the same morphology as any other. An analysis of the patterns of changes in morphology provides some insight into the genetic plan for the metameric body axis.

Animals↗

Genomic organization and expression of the CXCR4 gene in mouse and man: absence of a splice variant corresponding to mouse CXCR4-B in human tissues.

The human CXC chemokine receptor CXCR4 is activated by stromal cell-derived factor 1. The receptor is present in many cell types and regulates a variety of cellular functions, including chemotaxis, adhesion, hematopoiesis, and organogenesis. Human CXCR4 also serves as a cofactor for cell entry by certain strains of HIV-1 and HIV-2. In the mouse, alternative RNA splicing produces two transcripts encoding two CXCR4 isoforms, mCXCR4-A and mCXCR4-B, differing by the presence of two amino acids in the amino terminal portion of the longer protein, mCXCR4-B. Only one CXCR4 transcript, encoding the human counterpart of mCXCR4-A, is known in man. The involvement of the aminoterminal-most portion of CXCR4 in both ligand and HIV envelope protein recognition led us to determine whether a CXCR4 variant corresponding to mCXCR4-B is present in human tissues. To this end, the genomic organization and expression of the human CXCR4 gene was examined. Both the human and the mouse CXCR4 gene consist of two exons separated by an approximately 2.1 kbp intron between codons five and six and carry splice donor sites at the 5' end of their introns. These similarities notwithstanding, single nucleotide primer extension, reverse transcriptase PCR amplification, and sequencing of CXCR4 cDNA clones show that a splice variant of CXCR4 corresponding to mCXCR4-B is absent in man.

Amino Acid Sequence↗

Fibronectin isoform distribution in the mouse. II. Differential distribution of the alternatively spliced EIIIB, EIIIA, and V segments in the adult mouse.

The alternatively spliced EIIIB, EIIIA, and V segments of fibronectin (FN) show widespread codistribution in the mouse embryo, suggesting that EIIIB+, EIIIA+, and V+ isoforms serve to facilitate morphogenesis and organogenesis (Peters, JH, and Hynes, RO, 1996, this issue). To gain further clues to functions of these segments, we have used segment-specific anti-FN antibodies to perform immunofluorescence microscopy on tissue sections obtained from mice aged 9 to 15 weeks. Staining for each of the three spliced segments, relative to that for the total FN pool, was reduced in the adult as compared with the embryo. Anti-V antibodies produced patterns which were most similar to those obtained with anti-total FN antibodies, localizing to basement membranes, connective tissues subjacent to epithelia, walls of blood vessels, and cartilage. Anti-EIIIA antibodies produced the next most widespread pattern, which included prominent staining of the walls of blood vessels of all sizes, the lung interstitium, and smooth muscle associated with the gastrointestinal (GI), genitourinary (GU), and respiratory tracts. Although anti-EIIIB antibodies produced the faintest and most restricted pattern of staining, EIIIB+ FN could be detected in the walls of some smaller blood vessels, smooth muscle of the GI, GU, and respiratory tracts, as well as within cartilaginous structures, and eye. There were quantitative and/or qualitative differences in the staining patterns produced by the three segment-specific antibodies in a variety of tissues, including liver, cartilage, synovium, cornea, muscle, peripheral nerve, and lymph node. These findings suggest that each of the spliced segments of the FN molecule may occupy unique physical or functional positions within the extracellular matrix of the adult mouse.

Animals↗

Survival of rat or mouse ventral mesencephalon neurons after cotransplantation with rat sertoli cells in the mouse striatum.

Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.

Animals↗

Selecting interspecific human-mouse and Chinese hamster-mouse hybrids using a new half-selection technique with a polyene antibiotic.

Interspecific human-mouse and Chinese hamster-mouse hybrids were isolated from polyethylene glycol fused cells by a new half-selection technique employing a structurally modified polyene macrolide antibiotic, amphotericin B methyl ester (AME), and HAT media. Unfused parental cells were killed as a result of innate sensitivity to AME or their genetic deficiency, absence of thymidine kinase (TK-) or hypoxanthine guanine-phosphoribosyl transferase (HGPRT-). In contrast, hybrid colonies were isolated after two to three weeks growth in three or four changes of HAT-AME media and subsequent growth in HAT media alone. The ability of hybrid cells to proliferate using this selective protocol indicates that genetic complementation resulted, and polyene antibiotic resistance was expressed as a dominant phenotypic property in the hybrids. Hybrid selection was dependent on: (1) the number of cells of each parental cell type co-cultivated; (2) the level of polyene antibiotic administered; and (3) the time interval before selection was initiated. The half-selection technique described in this report is simple to use, very effective in eliminating unfused parental cells and increases the potential types of hybrids which can be formed. Only one parental cell type need contain a biochemical defect, whereas the second parental type can be genetically normal.

Amphotericin B↗

Antibiotic binding to human polymorphonuclear neutrophils, mouse leukemia L1210 cells and mouse thymocytes.

This report describes a system in which antibiotics could be compared for binding to different mammalian cells. These included functional phagocytes (human polymorphonuclear neutrophils; PMNs), non-phagocytic lymphocytes (mouse thymocytes), and non-functional leukocytes (mouse leukemia L1210 cells). When antibiotics bound to PMNs, they bound about the same to L1210 cells but much less to thymocytes. Combining these data with previous data, the ranking of cells that bound the greatest amount of antibiotics was: PMNs = L1210 cells = blood mononuclear leukocytes greater than thymocytes greater than erythrocytes. Thus, antibiotics bind differentially and not indiscriminately to mammalian cells.

Animals↗

Differential kappa-opioid receptor expression on mouse lymphocytes at varying stages of maturation and on mouse macrophages after selective elicitation.

The combination of indirect immunofluorescent labeling and flow cytometry has proven to be a sensitive method for labeling of the kappa-opioid receptor on mouse thymocytes. In the present study, this labeling procedure was applied, along with phenotypic analysis, to mature immune cell populations to determine whether kappa-opioid receptor expression is present after immune cell maturation. Unfixed primary splenocytes from 6- to 8-week-old C57BL/6ByJ male mice were incubated with the fluorescein-containing, kappa-selective ligand fluorescein-conjugated 2-(3, 4-dichlorophenyl)-N-methyl-N-[1-(3-aminophenyl)-2-(1-pyrrolidinyl)eth yl]acetamide (FITC-AA). Amplification of FITC-AA binding to the kappa-opioid receptor was attained by adding a biotin-conjugated antifluorescein antibody, followed by extravidin-R-phycoerythrin. It has been shown previously that greater than 60% of immature thymocytes (CD4(+)/CD8(+)) demonstrated specific kappa-opioid receptor labeling. However, the present report shows that less than 25% of either T-helper or T-cytotoxic splenic lymphocytes expressed the kappa-opioid receptor. Likewise, only 16% of all splenic B lymphocytes were labeled for the kappa-opioid receptor. These findings demonstrate a decrease in kappa-opioid receptor expression on maturation of mouse lymphocytes. Interestingly, resident peritoneal macrophages showed a greater magnitude of specific receptor labeling, compared with either thymocytes or splenocytes, and approximately 50% of the resting Mphi expressed the kappa-opioid receptor. However, elicitation of Mphi with thioglycollate resulted in the complete loss of the expression of this receptor. Taken together, these findings demonstrate the diversity in the expression of the kappa-opioid receptor on immune cells at varying stages of differentiation, with preferential expression demonstrated by resident, peritoneal macrophages.

Animals↗

Seminal vesicle autoantigen, a novel phospholipid-binding protein secreted from luminal epithelium of mouse seminal vesicle, exhibits the ability to suppress mouse sperm motility.

Seminal vesicle autoantigen (SVA) is a 19 kDa glycoprotein purified from mouse seminal vesicle secretion. It was quantified to be 0.9% (w/v) in the seminal vesicle fluid. We examined its distribution in the accessory sexual gland, characterized its binding sites on the sperm surface and assessed its effect on sperm motility. It was immunolocalized on the epithelium of the primary and secondary folds in the tissue. Mouse spermatozoa collected from caudal epididymis were devoid of SVA. A cytochemical study illustrated the presence of SVA-binding region on the entire cells. The cytochemical staining intensity for the binding of SVA to spermatozoa remained even when the cells were pretreated with protease digestion, acid or heat at 100 degrees C for 10 min. Moreover, the SVA-sperm binding could be inhibited by the dispersed sperm lipid. The specificity of interaction between (125)I-SVA and phospholipids was studied by TLC overlay techniques. The radiolabelled protein showed strong binding to purified phosphatidylcholine and phosphatidylserine and weak binding to purified sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine, but did not interact with phosphatidic acid, lysophosphatidic acid or phosphatidylinositol. Among the lipids extracted from spermatozoa, SVA showed strong binding to phosphatidylcholine and weak binding to sphingomyelin and neutral lipids. The assay for SVA-sperm binding with (125)I-SVA determined the IC(50) as being (3.89+/-0.65)x10(-5) M(-1), which is compatible with an apparent dissociation constant of (9.10+/-0.02)x10(-5) M(-1) estimated by fitting the data of phosphatidylcholine-perturbed SVA fluorescence to a modified Scatchard plot. SVA showed an ability to suppress sperm motility. The average path velocity, straight-line velocity and curvilinear velocity of sperm were not detectable by computer-assisted sperm assay after incubation of the cells in the presence of 0.3% SVA at 37 degrees C for more than 40 min.

Animals↗

[Adrenergic receptor and knockout mouse: 2). Alpha adrenergic receptor knockout mouse].

The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors which are known to have a critical role in regulating transmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system. alpha 2A adrenergic receptor knockout mouse (alpha 2A-KO) showed an increase in sympathetic activity with resting tachycardia, depletion of cardiac tissue norepinephrine concentration. Knockout mouse study reveals that the alpha 2B adrenoceptor (alpha 2B AR) mediates the hypertensive response, and the alpha 2A AR mediates the hypotensive response, induced by alpha 2 agonists. Both the alpha 2A and alpha 2C AR subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves. alpha 2A AR subtype inhibits transmitter release at high stimulation frequencies, whereas the alpha 2C AR subtype modulates neurotransmission at lower levels of nerve activity. Salt-induced hypertension experiment using alpha 2B-KO and alpha 2C-KO revealed that alpha 2B AR subtype is necessary to raise BP in response to dietary salt loading. Lack of adequately functional renal alpha 2B AR is thought to preclude reabsorption of sodium. alpha 1B-KO study revealed that alpha 1B AR is a mediator of the blood pressure response and the aorta contractile response induced by alpha 1 agonists.

Animals↗

Differentiation and malignant suppression induced by mouse erythroid differentiation and denucleation factor on mouse erythroleukemia cells.

OBJECTIVE: To investigate the roles of mouse erythroid differentiation and denucleation factor (MEDDF), a novel factor cloned in our laboratory recently, in erythroid terminal differentiation. METHODS: Mouse erythroleukemia (MEL) cells were transfected with eukaryotic expression plasmid pcDNA-MEDDF. Then we investigated the changes on characteristics of cell growth by analyzing cells growth rate, mitotic index and colony-forming rate in semi-solid medium. The expressions of c-myc and beta-globin genes were analysed by semi-quantitative RT-PCR. RESULTS: MEL cells transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index, and colony-forming rate in semi-solid medium (P<0.01). The percentage of benzidine-positive cells was 32.8% after transfection. The expression of beta-globin in cells transfected with pcDNA-MEDDF was 3.43 times higher than that of control (MEL transfected with blank vector, pcDNA3.1), and the expression of c-myc decreased by 66.3%. CONCLUSIONS: MEDDF can induce differentiation of MEL cell and suppress its malignancy.

Activins↗

[Differentiation and malignant suppression induced by mouse erythroid differentiation and denucleation factor on mouse erythroleukemia cells].

OBJECTIVE: To investigate the roles of mouse erythroid differentiation and denucleation factor (MEDDF), newly cloned in our laboratory, in erythroid terminal differentiation. METHODS: Mouse erythroleukemia cells (MEL) were transfected with eukaryotic expression plasmid pcDNA-MEDDF. The changes of cell growth rate, mitotic index and colony-forming rate in semi-solid medium were investigated. The expressions of c-myc and beta-globin genes were analysed by semi-quantitative RT-PCR. RESULTS: MEL cells transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index, and colony-forming rate in semisolid medium(P < 0.01). The percentage of benzidine-positive cells was 32.8% after transfection. The expression of beta-globin in cells transfected with pcDNA-MEDDF was 3.43 times higher than that of control (MEL transfected with blank vector, pcDNA3.1), and the expression of c-myc was decreased by 66.3%. CONCLUSIONS: MEDDF can induce differentiation of MEL cell, and suppress its malignancy likely.

Activins↗

Structure of the mouse IL-10 gene and chromosomal localization of the mouse and human genes.

The nucleotide sequence of a 7.2-kb segment containing the mouse IL-10 (mIL-10) gene was determined. Comparison to the mIL-10 cDNA sequence (Moore, K. W., et al. 1990. Science 248:1230; 250:494) revealed the presence of five exons that span approximately 5.1 kb of genomic DNA. The noncoding regions of the mIL-10 gene contain sequences that have been associated with transcriptional regulation of several cytokine genes. The mIL-10 gene was mapped to mouse chromosome 1 and the human IL-10 gene was also mapped to human chromosome 1.

Amino Acid Sequence↗

Functional analysis of the mouse alpha-fetoprotein enhancers and their subfragments in primary mouse hepatocyte cultures.

We have compared the activities of mouse alpha-fetoprotein (AFP) enhancers I, II, and III with their minimal enhancer fragments (Mers) I, II, and III and with the entire 7-kilobase pair enhancer domain by transient expression assay in primary fetal mouse liver cells. The level of expression directed by the AFP promoter [p(-1009)AFPcat] alone is stimulated at least 10-fold by the entire AFP enhancer domain (-1009 to -6983). Enhancer I can drive the level of chloramphenicol acetyltransferase activity equivalent to that of the entire enhancer domain, whereas the increase in activity by enhancers II and III is significantly lower (1.5-fold). MersI, II, and III all mediate a greater increase in activity than their corresponding enhancer regions. The increase with MerI is 16-fold. Using DNase I protection analyses we identified 3 protein-binding regions in MerI; site Ia binds liver and brain nuclear proteins; site Ib binds liver, kidney, and brain nuclear proteins as well as purified C/EBP; site Ic binds liver and kidney nuclear proteins. Site-specific mutation of Ia, Ib, or Ic showed a 10-25% reduction in chloramphenicol acetyltransferase expression; deletion of the C/EBP-binding site in Ib showed a 45% reduction in activity and mutation of all 3 sites (Ia, Ib, and Ic) resulted in a 75% reduction in activity. Our studies indicate no single trans-acting factor is absolutely essential for enhancer activity, and that the enhancer activity of MerI is mediated via a combinatorial and additive mechanism.

Animals↗

Mouse hepatitis virus infection suppresses modulation of mouse spleen T-cell activation.

Natural infection by mouse hepatitis virus (MHV) can affect interpretation of immunological studies in mice. MHV, a collective term describing a group of corona viruses, is found in natural infections in over 70% of laboratory mouse populations in the U.S.A. and Canada. Natural outbreaks of MHV in our animal colony afforded us the opportunity to study MHV-induced immunosuppression as well as the effects of MHV infection on neurotransmitter immunomodulation. Concanavalin A (Con A)-stimulated DNA synthesis by spleen T lymphocytes from MHV-infected mice was 20-50% that of non-infected mice. The MHV infection also altered neurotransmitter modulation of spleen T-lymphocyte activation. In contrast to noradrenaline ablation of Con A-activated DNA synthesis by spleen lymphocytes from non-infected mice, DNA synthesis by the infected group was not inhibited by noradrenaline or dibutyryl-cAMP. These effects of MHV infection were specific for spleen T lymphocytes since MHV infection did not alter Con A stimulation of thymocytes, lipopolysaccharide stimulation of spleen B lymphocytes, or noradrenaline inhibition of thymocyte and B-cell DNA synthesis. MHV infection also did not alter spleen T-lymphocyte subset proportions. Thus, MHV infection inhibits spleen T-lymphocyte activation and blocks in vitro catecholamine and cAMP regulation of spleen T-cell activation but does not affect activation of thymic cells or spleen B cells.

Animals↗

Stable expression of the mouse nicotinic acetylcholine receptor in mouse fibroblasts. Comparison of receptors in native and transfected cells.

A stable cell line expressing mouse acetylcholine receptors (AChRs), named AM4, was established by cotransfecting into NIH 3T3 fibroblasts, alpha-, beta-, gamma-, and delta-subunit cDNAs plus the neor gene by calcium phosphate precipitation. Surface AChRs on AM4 cells contain all four subunits, sediment as a single approximately 9 S peak on sucrose gradients, and have the same ratio of alpha- to beta-subunits as surface AChRs from mouse BC3H-1 cells. The surface AChRs exhibit pharmacological properties identical to those obtained for BC3H-1 cells, including the association and dissociation rates of alpha-bungarotoxin, a low affinity and cooperative instantaneous dose-response curve, cooperative steady state agonist binding and desensitization, cooperative enhancement of agonist binding affinity by local anesthetics, and distinct affinities for curariform antagonists. Patch clamp measurements on AM4 cells reveal AChR single channel properties identical to those obtained from BC3H-1 cells, including a single class of channels with a conductance of 56 pS, short and long duration openings at low and high agonist concentrations, brief and intermediate closed duration components at low agonist concentrations, and six distinct closed duration components at high agonist concentrations. The biochemical, pharmacological, and single channel measurements indicate at least 95% of the surface AChRs on AM4 cells are alpha 2 beta gamma delta pentamers.

Animals↗

Neoplastic transformation of mouse embryo cells by virus released from tumorigenic mouse cells transformed by avian sarcoma virus B77.

Mouse C3H embryo cells were transformed in vitro by avian sarcoma virus Bratislava 77 (B77) released scantily from a mouse cell line transformed earlier by the same virus. B77 virus transformed C3H embryo cells contained B77 viral genome and were transplantable into syngeneic as well as allogeneic DBA/2J young mice in which autochthonous sarcomas were induced. Tumors in both strains of mice were virogenic. The probable reasons for an increased transformation capacity of B77 virus in mammals are discussed.

Animals↗

Adverse effects of mouse hepatitis virus on ascites myeloma passage in the BALB/eJ mouse.

During experimental serial passage of ascites myelomas through BALB/cJ mice, unexpected illness and premature deaths occurred. Postmortem examination of affected mice revealed focal or diffuse discolored depressed areas in the liver and, in some cases, splenomegaly. Histopathologic findings consisted of focal to diffuse areas of necrosis with minimal leukocytic infiltration. Aerobic and anaerobic bacterial cultures of livers and spleens from affected mice were negative. Mouse hepatitis virus (MHV) was isolated from livers of clinically ill mice and from the ascites myeloma lines. An MHV contaminated ascites myeloma line, when passed into nude (nu/nu) mice, killed the animals in 6 days; the virus was isolated from livers of inoculated mice. Attempts to determine the source of the infection were unsuccessful. Serologic survey of newly acquired mice indicated no evidence of antibodies to MHV while mice in holding rooms had titers that ranged from 1:10 to 1:40. Two solid myeloma lines (being maintained by subcutaneous passage) were negative for MHV when tested by virus isolation techniques, and nine lines were negative to 11 murine viruses when tested by mouse antibody production assay. Attempts to demonstrate Eperythrozoon coccoides in control BALB/cJ mice were unsuccessful. Because of the outbreak, changes were made in animal handling procedures. A colony of BALB/cAn mice negative to MHV antibodies was established to provide animals for experimental passage of tumors, and animals in both the breeding and transfer room were placed under filter tops. The results were encouraging. In the four newly established tumor lines, one having been passed 46 times, no illness or unexplained deaths were observed.

Animals↗