H-2 influences phenytoin binding and inhibition of prostaglandin synthesis

We have reported that susceptibility to glucocorticoid- and phenytoin-induced cleft palate and glucocorticoid receptor levels in mice are influenced by the H-2 histocompatibility complex on chromosome 17. Phenytoin competes with glucocorticoids for the glucocorticoid receptor and inhibits production of prostaglandins and thromboxanes. In this paper we have investigated whether, as in the case of glucocorticoids, phenytoin receptor levels and phenytoin-induced inhibition of prostaglandins are influenced by H-2 in a variety of mouse tissues. Using congenic strains varying only in the H-2 region, but otherwise having either the A/Wy(A) or B10(B) genetic background, we demonstrate here that phenytoin receptor content in the lung and liver is significantly higher in the strains with H-2 ^a (A/Wy and B 10.A) than in their corresponding H-2 ^b partners (A.BY and B 10). The H-2 complex also influences phenytoin-induced inhibition of the release of ³H-arachidonic acid and prostaglandin biosynthesis from thymocytes, prelabeled with ³H-arachidonic acid. Thus, these results suggest a similar genetic and biochemical pathway for the teratogenic action of both phenytoin and glucocorticoids.     

Phenytoin teratogenicity in the primary and secondary mouse embryonic palate is influenced by the H-2 histocompatibility locus

Inbred and congenic strains of mice have been studied for susceptibility to phenytoin-induced cleft lip with or without cleft palate (CLP) and isolated cleft palate (CP). The role of genes linked to the H-2 complex on chromosome 17 has been confirmed. Congenic strains with the A background have identical levels of spontaneous CLP, whereas those strains having the A background with the H-2a haplotype have significantly higher rates of induced CLP than their congenic partners with the H-2b or H-2s haplotype. No such significant difference in the degree of CLP produced by phenytoin is demonstrable in strains with the B background. Rates of isolated CP produced by phenytoin are significantly higher in strains with H-2a than in their congenic partner strains with either H-2b or H-2s, whether the background is A or B.     

A histocompatibility region (H-2IC) in theIC region of theH-2 complex of the mouse

Skin graft rejection in congenic pairs of mice differing only at theH-2 complex appears to be influenced by at least 3 genes (H-2K, H-2D, H-2I); we now describe a fourth,H- 2IC: Grafts transplanted across anIC difference are sometimes rejected. TheI-C regions of three differentH-2 haplotypes (d,k,s) were studied in different combinations, and variable patterns emerged: (a)IC ^d : B10.S(7R) show delayed or no rejection of first B10.S(9R) grafts, but grafts to immunized recipients were usually rejected in 20 days; (b)IC ^k : in two combinations (A.AL A and B10.HTT B10.S[9R]) first grafts were rejected by day 30, although grafts to immunized mice showed a different pattern. In the third combination (B10.HTTB10.S[7R]) first grafts were retained but immunized mice rejected their grafts, (c)IC ^s : B10.S(9R) regularly reject B10.S(7R) first grafts, but immunized mice retain their grafts. In two other combinations first grafts were retained but grafts to immunized recipients were rejected; while in a third combination rejection did not occur at all. The background of the recipient appeared to be important in determining the variable pattern of rejection, and there is evidence for a similarity of the H-genes inIC ^s andIC ^k , and inIC ^k andIC ^p . Graft rejection occurred independently of known differences in Ia specificities, indicating thatH-2IC and the genes determining Ia specificities are probably different, although when grafts were performed in the presence of known la differences, graft rejection usually occurred.     

Presence of gamma-aminobutyric acid in embryonic palates of AJ and SWV mouse strains

The presence of gamma-aminobutyric acid (GABA) in the embryonic palate was sought as a criterion for its role in regulating palate development. GABA was measured by a gas chromatographic-mass spectrometric (GC-MS) method using the heptafluorobutyryl (HFB)-cyclohexyl-GABA derivative, which gave the necessary sensitivity and specificity to measure low levels of GABA in the presence of contaminating substances. GABA was measured in dissected embryonic palates at various times of development in the AJ mouse strain. GABA levels were lower in day 14 AJ palates (0.19 +/- 0.01 nmol/mg protein) than at days 13 (0.28 +/- 0.03) and 15 (0.30 +/- 0.04). Comparable levels were observed in fore- and hindlimbs at day 14, whereas levels were lower in embryonic tongue and higher, as was expected, in embryonic brain. To confirm the presence of GABA in the palate, it was analyzed in growing palate mesenchymal cells in primary and secondary cultures as well as in serum-free medium. In addition, GABA levels were compared in the SWV mouse strain; this strain exhibits a more efficient active uptake mechanism and diazepam produces a higher frequency of cleft palate in this strain than in AJ. SWV contained one and one-half to three times higher concentrations of GABA in excised palates and cultured palate cells than the AJ strain. Furthermore, when GABA levels in skin fibroblasts of the two strains were measured, SWV cells contained 2.7-fold greater GABA than AJ cells. The present results provide additional evidence for the role of GABA in palate development.     

Maternal phenytoin administration affects DNA and protein synthesis in embryonic primary palates

Recent studies have shown that phenytoin (Dilantin) administration to pregnant A/J mice on day 10 causes reduced growth in embryonic primary palates. The current investigation concentrates on biochemical and autoradiographic changes toward the end of primary palate formation (gestational day 11), which coincides with the developmental period used for the previously conducted morphological studies. On gestational day 10, one group of pregnant A/J mice was injected intraperitoneally (IP) with 60 mg/kg phenytoin and the other group with vehicle. Twenty-three hours after phenytoin administration, all animals were injected (IP) with either [3H]-thymidine or [3H]-leucine. After one hour of incorporation, animals were sacrificed, embryos removed and placed in ice-cold Eagle's minimum essential medium containing 0.02% NaN3 for biochemical assay or fixed immediately in Bouin's solution for autoradiography. For biochemical analyses, palates and limb buds were removed, homogenized, TCA precipitated, lyophilized, and acid hydrolyzed. Examination of the data revealed that DNA synthesis in control palates was 3.8-fold greater than in primary palates from embryos of phenytoin-treated mothers. Results were similar for limb buds from control embryos and from embryos of phenytoin-treated mothers. Experiments utilizing [3H]-leucine indicated that protein synthesis was 2.6-fold greater in primary palates from phenytoin-treated mothers than in control primary palates. Similar results were obtained for protein synthesis in limb-bud tissue from controls and embryos of phenytoin-treated mothers. Autoradiographic data supported the biochemical findings. DNA synthesis in primary palates from embryos of phenytoin-treated mothers decreased 3-fold; protein synthesis increased 2.2-fold compared with control primary palates.(ABSTRACT TRUNCATED AT 250 WORDS)     

H-2 histocompatibility antigens of subcellular membranes of mouse liver

Purified fractions of plasma membrane, Golgi apparatus, rough endoplasmic reticulum vesicles, nuclear envelope, and mitochondria were isolated from mouse liver and the distribution of H-2 histocompatibility antigens determined by indirect radioimmunoassay before and after membrane disruptive treatments. Fractions enriched in plasma membrane (surface membrane) revealed H-2 antigens in highest concentration; disruptive treatments were not necessary to reveal H-2 antigens with surface membranes. In contrast, internal membranes did not possess H-2 antigens which were accessible to antibody. Golgi apparatus fractions or some component of these fractions (e.g. secretory vesicles) possessed the antigens but in a latent form where accessibility was provided by simple rupture of the membrane vesicles. With endoplasmic reticulum, detergent solubilization of the membranes was required before H-2 antigen could be detected. Nuclear envelope preparations contained little or no demonstrable H-2 activity. These results were confirmed by several techniques including immunoprecipitation of labelled solubilized membrane components with anti-H-2 serum and subsequent analysis in SDS-PAGE.     

Dexamethasone-induced phospholipase A2-inhibitory proteins (PLIP) influenced by the H-2 histocompatibility region

Recent work has indicated that the H-2 histocompatibility complex on chromosome 17 influences the degree of glucocorticoid-induced teratogenicity and anti-inflammatory response. Since both of these hormonal actions appear to be mediated by the induction of phospholipase A2-inhibitory proteins (PLIP), the influence of the H-2 complex on the induction of PLIP by glucocorticoids in thymocytes and embryonic palates has been investigated. Analysis of dexamethasone-induced PLIP by Sephadex G-100 revealed four peaks of mol wt 55,000, 40,000, 28,000 and 15,000 in mouse thymocytes and from one to three of these PLIPs in mouse embryonic palates. The 55,000 mol wt PLIP comprised 50-60% of the total activity. The total amount of dexamethasone-induced PLIP is significantly higher in B10.A (H-2a) thymocytes than that in thymocytes of their congenic resistant partners, B10 (H-2b). The induced level of PLIP in the embryonic palates treated with dexamethasone is also significantly higher in the H-2a congenic strains with either the A or B background (AWy or B10.A) than that in their resistant partners (A.BY or B10). Thus, both susceptibility to glucocorticoid-induced cleft palate and the production of PLIP by this hormone are influenced by the H-2 complex.     

Molecular evolution of enzymes involved in the arachidonic acid cascade

The metabolic pathway called the arachidonic acid cascade produces a wide range of eicosanoids, such as prostaglandins, thromboxanes and leukotrienes with potent biological activities. Recombinant DNA techniques have made it possible to determine the nucleotide sequences of cDNAs and/or genomic structures for the enzymes involved in the pathway. Sequence comparison analyses of the accumulated sequence data have brought great insights into the structure, function and molecular evolution of the enzymes. This paper reviews the sequence comparison analyses of the enzymes involved in the arachidonic acid cascade.     

Immunocytochemical localization of class I H-2 histocompatibility antigens of mouse liver

The subcellular location of class I H-2 histocompatibility antigens was determined for mouse liver using immunocytochemical techniques and correlated with information determined by cell fractionation and analysis in situ. Surface antigens first were localized by standard procedures involving surface labeling with ferritin-labeled antibody. This approach could not be used for internal membranes either in situ or in fractions since the antigens are not expressed at the cytoplasmic surface. For this purpose, thin sections of tissues embedded in Lowicryl were analyzed and quantitated. The in situ analysis confirmed the presence of H-2 antigens on internal membrane compartments as well as on the cell surface and helped rule out the possibility that distributions based on analyses by immunoprecipitation of fractions of internal membranes were influenced greatly by plasma membrane contamination. Quantitation was provided by immunoprecipitation of H-2 antigens from radioiodinated or metabolically labeled isolated and highly purified cell fractions. The findings establish the presence of class I H-2 histocompatibility antigens in endoplasmic reticulum, Golgi apparatus and plasma membrane in the approximate ratios of 1:3:7. No class I H-2 histocompatibility antigens could be detected in mitochondria, salt extracts of isolated membranes or NP-40-insoluble membrane material.     

Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer

Among the more than 20 H-2-like genes in the BALB/c mouse genome, there are two classical transplantation antigens (H-2Dd and H-2Ld) encoded at the D-end of the major histocompatibility complex. Here we report the identification of a bacteriophage clone that encodes H-2Dd. The H-2Dd gene was identified by nucleotide sequence analysis and by characterization of the new H-2 antigen expressed when the cloned gene was introduced into mouse L cells by DNA-mediated gene transfer. The previously identified H-2Ld gene was then compared with the H-2Dd gene. The two genes appear to have the same general structure, and for the 854 nucleotides that have been compared, the two genes are 89% homologous. The H-2Ld and H-2Dd antigens expressed on mouse L cells after DNA-mediated gene transfer were examined by immunologic criteria. The stably transformed cell lines express apparently normal levels of H-2Dd and H-2Ld on the cell surface as measured by quantitative immunofluorescence by using monoclonal anti-H-2 antibodies. They synthesize H-2Dd and H-2Ld at normal rates as determined by endogenous labeling and immunoprecipitation of cell extracts. They evoke a strong specific serologic response when used to immunize C3H mice. The newly expressed antigens are able to serve as targets for alloreactive T cells. These cloned genes provide good substrates for examining the evolution of two closely linked H-2 antigen genes. Comparison of the structures of these genes provides clues to the basis for the differential expression of these antigens and their different biologic functions.     

Synthesis of a defective H-2 histocompatibility antigen by mutant mouse strain BALB/c-H-2 dm2

Mutant mouse strain BALB/c-H-2 ^dm2 (dm2), which fails to express the H-2L^d histocompatibility antigen associated with the wild type, BALB/c, synthesizes instead a smaller molecule that is structurally related to H-2L^d but does not carry detectable alloantigenic determinants. This new protein, p40, is a membrane glycoprotein found in dm2 cells but not in BALB/c. p40 was detected by electrophoresis of dm2 glycoprotein preparations and by immunoprecipitation with heterologous H-2-specific antibodies. The p40 molecule is found associated with intracellular membranes but was not detected at the cell surface. Peptide mapping studies suggest that dm2 carries an alteration in the H-2L ^d structural gene, which prevents proper maturation of the protein product.     

Graft vs host reaction in reciprocal combinations of mouse strains differing by the H-2 complex of histocompatibility]

Intraperitoneal transplantation of 0.5 x 10(7), 1 x 10(7) or 2 x 10(7) spleen cells from the C57BL mice to newborn CBA recipients induced an acute form or runt disease which resulted in the death of 43%, 86% or 95% of the recipient mice, respectively, in the course of 2--3 weeks after the cell transfer. Preliminary immunization of C57BL donors with CBA isoantigens led to a marked enhancement, and immunization with foreign antigens (sheep red blood cells)--to weakening the reaction. In reverse combination of mouse strains the runt disease was 4--5 times less severe and no "preimmunization effect" occurred. In C57BL leads to CBA combination the reaction was accompanied by proliferation of pyroninophilic mononuclears and follicular destruction, while in the CBA leads to C57BL combination-by the retardation of their growth.     

Vasoactive prostanoids are generated from arachidonic acid by COX-1 and COX-2 in the mouse

Generation of vasoactive prostanoids from arachidonic acid by cyclooxygenase (COX)-1 and COX-2 was investigated in anesthetized mice. Intravenous injections of the prostanoid precursor arachidonic acid increased pulmonary arterial pressure and decreased systemic arterial pressure. Pulmonary pressor and systemic depressor responses were attenuated by SC-560 and nimesulide, inhibitors of COX-1 and COX-2, in doses that did not alter responses to injected prostanoids. Pulmonary pressor responses to arachidonic acid were blocked and a depressor response was unmasked, whereas systemic depressor responses were not altered, by a thromboxane receptor antagonist. Pulmonary and systemic pressor responses to angiotensin II injections and systemic pressor responses to angiotensin II infusion were not modified by COX-1 or COX-2 inhibitors but were attenuated by losartan. Systemic depressor responses to arachidonic acid were smaller in COX-1 and COX-2 knockout mice, whereas responses to angiotensin II, norepinephrine, U-46619, endothelin-1, and PGE(1) were not different in COX-1 and COX-2 knockout and wild-type control mice. These results suggest that vasoactive prostanoids with pulmonary pressor and systemic vasodepressor activity are formed by COX-1 and COX-2 and are consistent with Western blot analysis and immunostaining showing the presence of COX-1 and COX-2. These data suggest that thromboxane A(2) (TxA(2)) is formed from the precursor by COX-1 and COX-2 in the lung and are in agreement with immunofluorescence studies showing thromboxane synthase. The present data suggest that COX-1- or COX-2-derived prostanoids do not modulate responses to angiotensin II or other vasoactive agents and that prostanoid responses are similar in CD-1 and C57BL/6 and in male and female mice.     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) were formed. A detailed analysis of cell culture supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography/masspectrometry revealed the presence of 5-, 8-, 12- and 15- monoHETE's, two distinct 5,12-diHETE's, several 8,15-diHETE's and 14,15-diHETE. Among the 5,12-diHETE's, only small amounts of a compound with the characteristics of LTB4 were detected. Under the conditions employed, the cyclooxygenase products PGE2 and PGI2 (as 6-keto-PGF1 alpha) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI2, PGE2 and LTC4 were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.     

Cell-free synthesis and membrane insertion of mouse H-2Dd histocompatibility antigen and beta 2-microglobulin.

Messenger RNA from SL2 lymphoma cells was translated in a cell-free system in the presence of microsomal membranes. Mouse H-2Dd histocompatibility antigen was correctly assembled in the microsomal membranes, and transmembrane insertion of the nascent chain was accompanied by glycosylation and cleavage of the signal sequence H-2Kd antigens, synthesized in vivo, comprised a transmembrane glycoprotein and an unglycosylated protein in the cytoplasm. The glycosylated forms of the H-2Dd and H-2Kd antigens were modified during intracellular transport from the endoplasmic reticulum to the cell surface. beta 2-Microglobulin was also synthesized in vitro, and transfer of this protein into microsomal vesicles was accompanied by cleavage of its signal sequence. In the endoplasmic reticulum, beta-microglobulin can bind to newly synthesized H-2d glycoproteins. The mRNAs coding for beta 2-microglobulin and H-2Dd antigen could be separated on aqueous sucrose gradients.     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography / masspectrometry revealed the presence of 5-, 8-, 12- and 15 - mono-HETE's, two distinct 5, 12-diHETE's, several 8, 15-diHETE's and 14, 15-diHETE. Among the 5, 12-diHETE's, only small amounts of a compound with the characteristics of LTB, were detected. Under the conditions employed, the cycloxygenase products PGE₂ and PGI₂ (as 6-keto-PGF_1g) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI₂, PGE₂ and LTC₄ were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.