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PubMed2001

Mapping and characterization of the mouse and human SS18 genes, two human SS18-like genes and a mouse Ss18 pseudogene.

We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus explaining the high and widespread expression of the SS18 genes.

Alternative Splicing↗
PubMed1995

Sequence of mouse Odf1 cDNA and its chromosomal localization: extension of the linkage group between human chromosome 8 and mouse chromosome 15.

The mouse cDNA encoding the major protein of the outer dense fibers in sperm tails was isolated by reverse transcription of testicular RNA and amplification with sequence-specific primers. Sequencing of a genomic clone obtained by inverse PCR yielded the 5' untranslated region. The transcription starting point was verified by primer extension. The putative proteins encoded by Odf1 in mouse and by ODF1 in rat and man are very similar. A total of 15 amino acids in the C-terminal region were deleted in the mouse protein, compared with the rat protein. Through in situ hybridization to metaphase chromosomes, the Odf1 gene was localized to mouse chromosome 15 region B2-C. The chromosomal localization of the Odf1 gene extends the hitherto known linkage group consisting of MYC (Myc), PVT1 (Pvt1), GPT (Gpt), and TG (Tg) common to human chromosome 8 and mouse chromosome 15 in the proximal direction of both chromosomes. The linkage group now extends from band q24 to band q22 of human chromosome 8 and from region D2-E to region B2-C of mouse chromosome 15.

Amino Acid Sequence↗
PubMed1996

Genetic mapping of the mouse stromal cell-derived factor gene (Sdf1) to mouse and rat chromosomes.

Stromal cell-derived factor 1 (SDF1) is a new member of the Cys-X-Cys chemokine family. The chromosomal location of Sdf1, the gene coding mouse SDF1, was determined by fluorescence in situ hybridization (FISH) and molecular linkage analysis. The mouse Sdf1 gene was localized to the R-band-positive F1 band of chromosome 6 by direct R-banding FISH. Interspecific backcross analysis identified the mouse Sdf1 gene locus at 0.8 cM terminal to D6Nit55 and 3.0 cM proximal to D6Mit12. With in situ hybridization using a mouse cDNA clone as a probe, the rat Sdf1 gene was localized to the R-band-positive band 4q42.1, where conserved linkage homology to mouse chromosome 6 has been identified. Although other Cys-X-Cys chemokine genes have been mapped on human chromosome 4, the chromosomal segment where the mouse and rat Sdf1 gene reside have no conserved linkage homology to human chromosome 4. This result suggests that SDF1 is a new chemokine class.

Amino Acid Sequence↗
PubMed2005

Electrophysiological characterization of pancreatic islet cells in the mouse insulin promoter-green fluorescent protein mouse.

We recently reported a transgenic [mouse insulin promoter (MIP)-green fluorescent protein (GFP)] mouse in which GFP expression is targeted to the pancreatic islet beta-cells to enable convenient identification of beta-cells as green cells. The GFP-expressing beta-cells of the MIP-GFP mouse were functionally indistinguishable from beta-cells of normal mice. Here we characterized the ionic channel properties and exocytosis of MIP-GFP mouse islet beta- and alpha-cells. Beta-cells displayed delayed rectifying K+ and high-voltage-activated Ca2+ channels and exhibited Na+ currents only at hyperpolarized holding potential. Alpha-cells were nongreen and had both A-type and delayed rectifier K+ channels, both low-voltage-activated and high-voltage-activated Ca2+ channels, and displayed Na+ currents readily at -70 mV holding potential. Alpha-cells had ATP-sensitive K+ channel (KATP) channel density as high as that in beta-cells, and, surprisingly, alpha-cell KATP channels were more sensitive to ATP inhibition (IC50=0.16+/-0.03 mM) than beta-cell KATP channels (IC50=0.86+/-0.10 mM). Whereas alpha-cells were rather uniform in size [2-4.5 picofarad (pF)], beta-cells varied vastly in size (2-12 pF). Of note, small beta-cells (<4.5 pF) showed little exocytosis, whereas medium beta-cells (5-8 pF) exhibited vigorous exocytosis, but large beta-cells (>8 pF) had weaker exocytosis. We found no correlation between beta-cell size and their Ca2+ channel density, suggesting that Ca2+ influx may not be the cause of the heterogeneity in exocytotic responses. The MIP-GFP mouse therefore offers potential to further explore the functional heterogeneity in beta-cells of different sizes. The MIP-GFP mouse islet is therefore a reliable model to efficiently examine alpha-cell and beta-cell physiology and should greatly facilitate examination of their pathophysiology when the MIP-GFP mice are crossed with diabetic models.

Adenosine Triphosphate↗
PubMed1992

Development and validation of a radioimmunoassay for mouse osteocalcin: paradoxical response in the Hyp mouse.

The hypophosphatemic (Hyp) mouse is a model for human familial hypophosphatemic rickets. To test the hypothesis that there is an osteoblastic defect in these animals, serum osteocalcin levels were measured in Hyp mice and their normal littermates. Furthermore, the effects of phosphorus deprivation, phosphorus loading, and 1,25-dihydroxyvitamin D3 administration on serum osteocalcin levels were examined. Osteocalcin was purified from mouse hindlimbs, and a polyclonal antibody to this material was produced in a goat. The antibody recognized native and decarboxylated mouse osteocalcin, but could not recognize osteocalcin from several other species. A RIA was developed which had a minimal detection limit of 0.4 nmol/liter (2.2 micrograms/liter) and half-maximal displacement at 2.7-3.3 nmol/liter (14.8-18.2 micrograms/liter). The intraassay coefficient of variation was 6.4%, while the interassay coefficient of variation was 12%. Dilutions of mouse serum samples varied by less than 15%. Analytical recovery was typically greater than 90%. Serum osteocalcin concentrations in Hyp and normal mice were shown to decrease with age. However, circulating osteocalcin levels in Hyp mice were higher than those in their normal littermates regardless of the age of the animal (P less than 0.001). One week of a high phosphorus diet resulted in an increase in serum phosphate in normal and Hyp mice, but serum osteocalcin concentrations were unaffected. On the other hand, dietary phosphorus deprivation for 4 weeks resulted in comparable hypophosphatemia in both Hyp and normal mice, and serum osteocalcin increased in both groups of animals. Intraperitoneal injection of 30 ng/day 1,25-dihydroxyvitamin D3 for 7 days resulted in a 215 +/- 33% increase in serum osteocalcin in normal animals, while the same regimen produced a 250 +/- 29% decrease in the Hyp mouse. Our results are consistent with the hypothesis that abnormal osteoblastic activity is present in Hyp mice. Furthermore, hypophosphatemia may be a general regulator of osteocalcin synthesis or secretion in the mouse.

Animals↗
PubMed1991

Isolation and characterization of mouse complementary DNAs encoding alpha and beta thyroid hormone receptors from thyrotrope cells: the mouse pituitary-specific beta 2 isoform differs at the amino terminus from the corresponding species from rat pituitary tumor cells.

Thyroid hormones (T3) and their receptors (TR) play a critical role in the function of the pituitary gland, particularly in thyrotropes, where they regulate expression of the alpha- and beta-subunits of TSH. Since the pituitary gland is composed of several cell types, we undertook a characterization of TR subtypes in a murine thyrotropic tumor (TtT-97), an excellent model in which to study thyroid hormone action in thyrotropes. We screened a thyrotrope cDNA library with rat TR alpha 1 and TR beta 1 cDNA probes and isolated cDNAs encoding the mouse TR alpha 1 and TR beta 1 isoforms as well as a partial clone corresponding to the non-T3 binding carboxy-terminal alpha 2 variant. The polymerase chain reaction was used to amplify additional cDNAs for the specific 5' domains of the mouse TR beta 1 and the pituitary-specific TR beta 2 amino-terminal variant. Using hybridization probes that discriminate between the alpha and beta isoforms and their variants, we demonstrated that thyrotropes contain TR alpha 1 and alpha 2 mRNAs as well as transcripts encoding Rev-erbA, which arise by transcription from the opposite strand of the TR alpha gene. In thyrotropes, the ratio of alpha 2 to TR alpha 1 mRNA levels more closely resembled the distribution in mouse brain than that in heart, where the mRNA levels of TR alpha 1 and alpha 2 are comparable. TR beta 1 and TR beta 2 mRNAs were detected in thyrotropes and were of similar size (approximately 6.4 kilobases). Despite the almost complete conservation between the rat and mouse TR beta 1 sequences at the protein level, the mouse and rat TR beta 2-specific N-terminal domains were less conserved, and the mouse protein was shorter by 39 amino acids at the N-terminus. Of the receptor species, only the mRNA encoding the TR beta 2 isoform, which was restricted to thyrotropes, was decreased by T3 treatment, although the mRNA for the alpha 2 variant was also reduced by T3 in thyrotropes and heart tissue. Levels of TR beta 1 mRNA were not changed in liver, but were increased in thyrotropic tumors and also somewhat in brain, an organ that is not responsive to T3 by classical criteria.

Amino Acid Sequence↗
PubMed2006

Application of laser-assisted zona drilling to in vitro fertilization of cryopreserved mouse oocytes with spermatozoa from a subfertile transgenic mouse.

Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.

Animals↗
PubMed2006

Conditional expression of human 15-lipoxygenase-1 in mouse prostate induces prostatic intraepithelial neoplasia: the FLiMP mouse model.

The incidence and mortality of prostate cancer (PCa) vary greatly in different geographic regions, for which lifestyle factors, such as dietary fat intake, have been implicated. Human 15-lipoxygenase-1 (h15-LO-1), which metabolizes polyunsaturated fatty acids, is a highly regulated, tissue-specific, lipid-peroxidating enzyme that functions in physiological membrane remodeling and in the pathogenesis of atherosclerosis, inflammation, and carcinogenesis. We have shown that aberrant overexpression of 15-LO-1 occurs in human PCa, particularly high-grade PCa, and in high-grade prostatic intraepithelial neoplasia (HGPIN), and that the murine orthologue is increased in SV40-based genetically engineered mouse (GEM) models of PCa, such as LADY and TRansgenic Adenocarcinoma of Mouse Prostate. To further define the role of 15-LO-1 in prostate carcinogenesis, we established a novel GEM model with targeted overexpression of h15-LO-1 in the prostate [human fifteen lipoxygenase-1 in mouse prostate (FLiMP)]. We used a Cre- mediated and a loxP-mediated recombination strategy to target h15-LO-1 specifically to the prostate of C57BL/6 mice. Wild-type (wt), FLiMP+/-, and FLiMP+/+ mice aged 7 to 21, 24 to 28, and 35 weeks were characterized by histopathology, immunohistochemistry (IHC), and DNA/RNA and enzyme analyses. Compared to wt mice, h15-LO-1 enzyme activity was increased similarly in both homozygous FLiMP+/+ and hemizygous FLiMP+/- prostates. Dorsolateral and ventral prostates of FLiMP mice showed focal and progressive epithelial hyperplasia with nuclear atypia, indicative of the definition of mouse prostatic intraepithelial neoplasia (mPIN) according to the National Cancer Institute. These foci showed increased proliferation by Ki-67 IHC. No progression to invasive PCa was noted up to 35 weeks. By IHC, h15-LO-1 expression was limited to luminal epithelial cells, with increased expression in mPIN foci (similar to human HGPIN). In summary, targeted overexpression of h15-LO-1 (a gene overexpressed in human PCa and HGPIN) to mouse prostate is sufficient to promote epithelial proliferation and mPIN development. These results support 15-LO-1 as having a role in prostate tumor initiation and as an early target for dietary or other prevention strategies. The FLiMP mouse model should also be useful in crosses with other GEM models to further define the combinations of molecular alterations necessary for PCa progression.

Animals↗
PubMed1976

Competitive inhibition of passive sensitization of mouse mast cells by IgE. A bioassay for mouse and rat IgE.

Possibility of inhibition of an efficient in vitro IgE-sensitization system was studied. The sentization of mouse peritoneal mast cells with an anti-ovalbumin IgE-rich fraction of serum, as tested by ovalbumin-induced degranulation, was inhibited by previous incubation with antisera of another or of no specificity. Fractionation and other experiments showed that the inhibiting activity correlated with IgE content. IgGl did not seem to have an effect. Sensitization was also inhibited by rat myeloma IgE, 50 ng giving a 50 per cent inhibition. Plots of the logarithms of rat and mouse IgE concentration vs their inhibitory effect on sensitization gave two parallel linear curves, indicating that mouse and rat IgE compete for the same receptor sites. It was thus possible to use this system as a sensitive bioassay for both mouse and rat IgE levels and, by comparing inhibition by mouse IgE to that by a known rat IgE standard, to obtain not only relative data but absolute mouse IgE levels. This, and also a better discrimination of IgE doses, was the major advantage of this bioassay in relation to the equally sensitive anti-IgE degranulation tests.

Animals↗
PubMed2000

Effect of mouse strain and age on detection of mouse parvovirus 1 by use of serologic testing and polymerase chain reaction analysis.

BACKGROUND AND PURPOSE: Detection of mouse parvovirus 1 (MPV) depends on use of serologic and polymerase chain reaction (PCR) assays. These assays were evaluated for their ability to detect virus-specific antibodies or viral DNA in multiple strains and ages of mice inoculated with MPV. METHODS: Twelve-week-old ICR, BALB/c, C3H, C57BL/6, and DBA/2 mice and four- and eight-week-old ICR mice were inoculated with MPV. Serum was harvested four weeks after inoculation and analyzed by use of recombinant non structural protein 1 (rNS1) enzyme-linked immunosorbent assay (ELISA), minute virus of mice (MVM) ELISA, and MPV indirect fluorescent antibody (IFA), MVM IFA, and MPV hemagglutination inhibition (HAI) assays. Select tissues were harvested and analyzed by use of an MPV-specific PCR assay. RESULTS: The number of mice in each group with detectable MPV-specific antibodies or MPV DNA varied with mouse strain, mouse age when inoculated, and viral dose. Seroconversion in mice inoculated at 12 weeks of age was detected almost exclusively by use of the MPV IFA and MPV HAI assays, whereas seroconversion in almost all mice inoculated at 4 and 8 weeks of age was detected by use of all immunoassays except the MVM ELISA. Viral DNA was detected by use of PCR analysis in all strains and ages of mice except DBA/2 mice. CONCLUSIONS: Mouse strain and age have important roles in seroconversion to nonstructural and structural MPV antigens and persistence of viral DNA in mouse tissues. Therefore, diagnostic serologic testing and PCR analysis should be considered within the context of mouse strain and age at the time of MPV exposure, especially when sentinel mice are used for surveillance.

Animals↗
PubMed2003

Mouse tales from Kresge: the deafness mouse.

Mouse models for human deafness have not only proven instrumental in the identification of genes for hereditary hearing loss, but are excellent model systems in which to examine gene function as well as the resulting pathophysiology. One mouse model for human nonsyndromic deafness is the deafness (dn) mouse, a spontaneous mutation in the curly-tail (ct) stock. The dn gene is on mouse Chromosome 19 and it was recently shown to be a novel gene called Tmc1. A mutation in Tmc1 is also found in Beethoven (Bth), which is another deaf mouse mutant. In humans, one autosomal dominant form of nonsyndromic hearing loss (DFNA36) and two autosomal recessive forms (DFNB7 and DFNB11) are associated with mutations in TMC1, the human homologue of Tmc1. The transmembrane protein encoded by this gene is required for normal cochlear hair cell function and the mouse models will facilitate the elucidation of the molecular pathway that is disrupted when mutations are present.

Animals↗
PubMed1991

Kinetics of early T-cell repopulation in fully xenogeneic chimeras (F344 rat----B10 mouse): evidence for rat T-cell maturation in a xenogeneic mouse thymus.

We recently reported the model of fully xenogeneic chimerism achieved by transplantation of rat bone marrow into mouse recipients (F344 rat----B10 mouse), resulting in stable long-term rat lymphoid chimerism. We have now extended this model to examine whether developing precursor rat T cells from rat bone marrow stem cells can undergo normal differentiation in mature lymphocytes under the influence of a xenogeneic mouse thymus. We examined thymic and splenic lymphoid cells from fully xenogeneic chimeras starting 1 week after bone marrow transplantation to characterize early T-cell repopulation and phenotype. Our data suggest that developing rat precursor T cells are able to undergo normal differentiation in the mouse thymus. The first precursor T cells appeared 2 weeks after reconstitution and by week 10 accounted for more than 90% of thymocytes present in the chimeras. In chimeras, developing rat T lymphocytes in the mouse thymus exhibited an immature pattern (Thy 1.1+, alpha beta-TCRdull, CD4+ plus CD8+) when analyzed by flow cytometry. This pattern was similar to a normal rat. In contrast, splenic T-lymphoid cells showed a mature rat phenotype (Thy 1.1-, alpha beta-TCRhi, CD4+ or CD8+), again similar to a normal rat. This development began 2 weeks after bone marrow transplantation, and both thymus and spleen from chimeras exhibited "normal" rat T-cell staining profiles by 10 weeks after reconstitution. Overall, these data indicate that developing rat T cells are capable of undergoing normal maturation in a xenogeneic mouse thymus of tolerant animals.

Animals↗
PubMed1989

Transcriptional regulation of ferritin H and L subunits in adult erythroid and liver cells from the mouse. Unambiguous identification of mouse ferritin subunits and in vitro formation of the ferritin shells.

Ferritin H and L subunits present cell-specific features of structure, function, and transcriptional regulation. Mouse Friend erythroleukemia cells offer an interesting model to analyze the erythroid-specific expression of ferritin genes for comparison with the liver, an iron-storing tissue. cDNA clones for mouse ferritin H and L subunits have been isolated and sequenced. The two subunits have very similar calculated masses, 20.9 and 20.6 kDa for H and L, respectively. Electrophoretic analysis of the subunits encoded by the cDNA 1) allows unambiguous identification of mouse ferritin subunits; 2) clearly shows that mouse H and L chains can make heteropolymers in vitro; and 3) demonstrates that, at least in vitro, free subunits can coexist with subunits polymerized into complete shells. The mouse ferritin gene family displays a variable degree of complexity, ranging from three homologous sequences for the H genes to 10-14 homologous loci for the L genes. Transcription of ferritin genes exhibits tissue-specific difference. Nuclear transcriptional run-off experiments show that the L gene is more actively transcribed in the liver than in Friend erythroleukemia cells at different stages of maturation. The accumulation of the H subunit mRNA which results from dimethyl sulfoxide induction of Friend cells is the consequence of an increase in the transcription rate of the H gene. However, the H gene mRNA is transcribed at a similar rate in the liver and in induced Friend cells although 5-fold more mRNA accumulates in these cells. Therefore, there is a tissue-specific regulation of mouse ferritin expression at both the transcription and mRNA stability levels.

Animals↗
PubMed1987

Tumorigenicity in the nude mouse of cocultures derived from two nontumorigenic cell types, human pituitary adenomas and mouse C3H 10T1/2 fibroblasts.

Human pituitary adenoma tissues were not tumorigenic in the hormonally manipulated nude mouse. Mouse fibroblast cells (C3H 10T1/2) also did not form tumors when inoculated alone into nude mice. When these two tissues were cocultured and coinoculated into nude mice however, the majority of inocula developed progressively enlarging tumors which could be established in tissue culture and passaged through the nude mouse. These tumors were sarcomatous histologically and thus did not resemble any human pituitary adenoma tissue injected. In order to detect any human cells in these tumors, tumor genomic DNA was subjected to Southern analysis using human repetitive Alu and HGH DNA sequence probes. Southern blot analysis of the nude mouse derived tumor genomic DNA revealed no sizeable human DNA in the mouse tumor cell genome indicating the absence of significant numbers of human cells in the tumors or the transfer of human DNA to the mouse cells. The tumors therefore arose from transformed C3H 10T1/2 cells after coculture with the human pituitary adenoma cells. These results implied that the tumorigenic transformation of susceptible C3H 10T1/2 cells in the cocultures occurred as a result of the secretion by the adenoma cells of transforming substances in the culture media or the induction of tumorigenicity through direct cell-cell contact between the two cell types.

Adenoma↗
PubMed1987

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. VI. Detection of a non-MHC gene(s) in the E alpha-bearing RIIIS mouse strain that is associated with a specific lack of T cell responses to the M. arthritidis soluble mitogen.

Previous work using inbred, congenic and recombinant mouse strains showed a positive association with expression of E alpha and the ability of splenic cells to bind to and undergo proliferation in response to a T cell mitogen present in culture supernatants of Mycoplasma arthritidis (MAS). Studies described in the present manuscript confirm this association because lymphocytes from mice expressing H-2a, H-2d, H-2j, H-2k, H-2p, H-2u, and H-2v all of which possess E alpha responded to MAS, whereas those expressing H-2b, H-2f, H-2q, and H-2s, which lack E alpha, failed to respond. One exception was noted in that the inbred RIIIS mouse (H-2r) that expresses E alpha failed to respond to MAS but responded normally to concanavalin A, and phytohemagglutinin. In contrast, the congenic B10.RIII (H-2r) mouse did respond to MAS, suggesting the presence of an MAS nonresponsive, non-major histocompatibility complex (MHC) gene(s) in the RIIIS mouse. MAS nonresponsiveness in the RIIIS mouse was recessive because the lymphocytes from F1 crosses with responder B10.RIII (H2r) and C3H (H2k) mice responded to MAS. Analysis of (RIIIS X B10.RIII)F1 X RIIIS or B10.RIII parental test cross progeny confirmed that nonresponsiveness to MAS was associated with a recessive, non-MHC gene(s). Evidence was also found that a non-MHC, MAS-nonresponsive gene(s) is also present in the inbred SWR (H-2q) and SJL (H-2s) strains, because lymphocytes from F1 crosses between these strains and the RIIIS mouse failed to respond to MAS. Both RIIIS and B10.RIII splenic cells bound the mitogen in MAS to a similar degree, confirming the presence of the binding site in both mice. In contrast, C3H.SW (H-2b) splenic cells that do not express E alpha failed to bind the mitogen. The nonresponsiveness of RIIIS lymphocytes to MAS was exercised at the level of the T cell rather than the accessory cell. Thus RIIIS T cells failed to respond to MAS presented by RIIIS, B10.RIII, or (RIIIS X B10.RIII)F1 accessory cells. In contrast, B10.RIII and (RIIIS X B10.RIII)F1 T cells responded to MAS when presented by RIIIS, B10.RIII, or F1 accessory cells. Similar observations were made using SWR and SJL T cells, which failed to respond to MAS irrespective of the source of accessory cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

The 65-kDa phorbol-diester hydrolase in mouse plasma is esterase 1 and is immunologically distinct from the 56-kDa phorbol-diester hydrolase in mouse liver.

Esterase 1, a well-characterized mouse plasma protein of unknown function, has activity against a wide range of ester substrates including beta-alanine nitrophenyl esters and 17 beta-esters of estradiol. In this article, we report that esterase 1 is also responsible for a majority of the phorbol-12-ester hydrolase activity in mouse plasma. Incubation of homogeneous esterase 1 with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) at either 4 or 37 degrees C for up to 18 h yielded phorbol 13 alpha-acetate as the only hydrolysis product. Specific polyclonal antibodies to esterase 1 inhibited 95% of PMA hydrolysis by a purified esterase 1 preparation and 65% of PMA hydrolysis by mouse plasma. Perfused mouse liver homogenates contain two distinct phorbol diester hydrolases with apparent molecular masses of 65 kDa and 56 kDa, respectively. The 65-kDa protein appears to be immunologically identical to the plasma enzyme, while the 56-kDa protein, found in liver but not in plasma, is immunologically distinct. Phorbol 12-myristate, phorbol 12,13-dibutyrate, and PMA were found to be competitive inhibitors of the beta-alanine-nitrophenyl esterase activity of esterase 1 with Ki values of approximately 7 microM. Phorbol 13-acetate and phorbol itself were less effective with Ki values of 37 and 140 microM, respectively. Sodium salts of valeric and myristic acids did not inhibit at 10 microM. The above results indicate that efficient substrate binding requires a phorbol 12-ester. Similar results were obtained with estradiol 17 beta-valerate which is a better substrate for esterase 1 than is PMA. Our results strongly suggest that esterase 1 and a recently described phorbol ester hydrolase isolated from mouse serum (Saito, M., and Egawa, K. (1984) J. Biol. Chem. 259, 5821-5826) are the same and are immunologically and kinetically distinct from the 56-kDa phorbol 12-ester hydrolase in mouse liver.

Animals↗
PubMed1981

Serum-mediated suppression of nonspecific B cell activation. II. Relative resistance of B cells from the NZB mouse strain to regulation by a natural inhibitor in normal mouse serum.

Recent work has shown that normal mouse serum (NMS) and plasma contain inhibitory substance(s), termed NMS-In, that suppress proliferative and polyclonal antibody responses elicited in vitro with various B cell mitogens. In this study, the NZB mouse, which has a high degree of spontaneous polyclonal B cell activity, was examined for possible reduced amounts of NMS-In. Also, the capacity of NZB B cells to be inhibited by NMS-In was determined. It was found that sera from NZB mice had normal amounts of NMS-In, as assessed by inhibition of mitogenesis of spleen cells in culture induced by bacterial endotoxin (ET). However, B cells of the NZB mouse were found to be abnormal, requiring approximately 4 to 7 times more NMS-In to inhibit polyclonal antibody synthesis and mitogenesis elicited by ET than B cells of more immunologically normal mice. Removal of T cells by treatment with anti-thy 1.2 and complement did not change the sensitivity of the NZB B cells to NMS-In. Insensitivity to NMS-In was expressed in B cells from relatively young NZB mice, e.g., 9 to 15 wk of age. The relative resistance of B cells of the NZB mouse to regulation by a natural inhibitor in NMS could partially serve to explain their high level of spontaneous antibody synthesis. Furthermore, the fact that B cells of the NZB mouse were ultimately susceptible to inhibition by relatively large amounts of NMS-In affords the opportunity in the future to suppress polyclonal antibody activity in the NZB mouse with purified NMS-In in an attempt to ameliorate autoimmune disease.

Animals↗
PubMedDate not supplied

Metabolism of mouse growth hormone-releasing factor, mGRF(1-42)OH, and selected analogs from the bovine GRF series in mouse and bovine plasma in vitro.

The presence of Val2 in mGRF(1-42)OH is unique and, as shown in this study, renders this GRF resistant to plasma DPP-IV, the main enzyme responsible for rapid hydrolysis and inactivation of Ala2-containing GRFs from other species via cleavages between Ala2-Asp3. The presence of DPP-IV activity in mouse serum, and mouse and bovine plasma has been demonstrated with Gly-Pro-p-nitroanilide and/or with two DPP-IV-sensitive bGRF analogs, [Leu27]bGRF(1-29)NH2 and [Ala15,Leu27]bGRF(1-29)NH2, which were effectively converted to their respective (3-29) fragments. During incubations of mGRF(1-42)OH in mouse serum or plasma, as well as in bovine plasma in vitro, no major fragments were detectable, except for small amounts of metabolites with HPLC retention times corresponding to those of mGRF(12-42)OH and mGRF(21-42)OH, indicative of possible trypsin-like cleavages between Arg11-Lys12 and Arg20-Lys21. Both mGRF(1-42)OH (t1/2 52-78.5 min) and [Val2,Ala15,Leu27]-bGRF(1-29)NH2 (t1/2 78.5 min) disappeared 5 to 7 times faster in mouse than in bovine plasma, indicating much higher activity of various degrading enzymes in mouse plasma. In summary, our data provide evidence that mGRF(1-42)OH, despite its resistance to plasma DPP-IV, is degraded relatively fast in mouse plasma or serum because of trypsin-like and other, non-DPP-IV-related, proteolytic cleavages.

Amino Acid Sequence↗