Cross-induction of predominant NPb idiotypic antibodies with derivatives of (4-hydroxy-3-nitrophenyl) acetyl

Immunization of C57BL/6 mice with bovine gamma-globulin (BGG) conjugated with (4-hydroxy-3-nitrophenyl) acetyl (NP) induced a population of anti-NP antibodies that bear predominantly lambda light chain, exhibit heteroclitic affinity for heterologous NP derivatives, and share NPb idiotype. The present study analyzes the idiotypes of antibodies induced with BGG conjugated with the iodo-, bromo-, or nitro-NP derivatives (NIP, NBrP, and NNP). NIP-BGG, NBrP-BGG, and NNP-BGG, induce specific antibodies bearing NPb idiotype in C57BL/6 or C57BL/10 congenic mice, but not in many other inbred strains. Furthermore, the quantity of NPb idiotype in immune sera from various mouse strains immunized with NIP-BGG, NBrP-BGG, and NNP-BGG was similar to that in sera from mice immunized with NP-BGG. Anti-idiotypic antisera against C57BL anti-NP, anti-NIP, or anti-NBrP antibodies exhibit extensive idiotype binding of specifically purified B6 anti-NP, -NIP, -NBrP, and -NNP antibodies. These purified antibodies contain a high percentage (greater than 70%) of lambda-chain-bearing molecules. The data indicate that an extensively shared repertoire composed of predominantly lambda-bearing NPb-positive idiotypic antibodies is used in response to NP and its derivatives in C57BL mice.     

A cross-over chromosome combiningIg heavy chain genes of two mouse strains

Two congenic strains of mice were bred in Konstanz that bear theIg heavy chain allotype gene of the C57BL/6 (Ig-1 ^b ) in a BALB/c background genome. One line (CB-8K) underwent eight backcross generations to BALB/c before sister-brother mating was initiated. For the other line (CB-16KN) backcrossing was continued for eight further generations, then a homozygousIg-1 ^b /Ig-1 ^b strain was produced by sister-brother mating. Both lines were tested for four V_H markers of the BALB/c and one of the C57BL parent. The CB-16KN strain expressed the C57BL marker V_HNP^b together with the C57BL allotype marker, and failed to express the three BALB/c markers, V_HDEX, V_HS117, V_HphOx and V_HNP^a. It thus resembled the CB-20 strain.Strain CB-8K expressed the V_HNP^b marker of C57BL but also all the four BALB/c markers that were tested. The strain appeared more heterogeneous than the CB-16KN strain, and a subline was bred from two exceptional mice that did not express the V_HNP^b marker. This subline (CB-8KN) expressed the BALB/c marker V_HNP^a regularly, and was negative for the V_HNP^b marker. It thus resembles the BAB-14 line.The crossing over event thus must have happened in one of the two meioses, which led to the CB-8K line. As BAB-14 is derived in an analogous manner to a branch of the backcross of Potter and Lieberman, which ended up in CB-20, the unexpected finding is discussed that two independent crossing over events (in CB-8KN and BAB 14) within theIg heavy chain gene region have taken place at approximately the same stage of two breeding programs.Recipient of an EMBO fellowship during part of the study.     

Cloning of mitogen- and antigen-reactive B lymphocytes on filter paper discs. II. Paratope frequencies within the mitogen-selected repertoire

Paratopic frequencies of C57BL/6 (Igh-Vb) and BALB/c (Igh-Va) mice were compared by determining the frequency of lipopolysaccharide-reactive, splenic B lymphocytes secreting antibody specific for (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP), trinitrophenyl (TNP), phosphorylcholine (PC), NIP/TNP, NIP/PC, and sheep erythrocytes. Despite the known genotypic and phenotypic differences between the two Igh-V loci, no significant differences in paratope frequencies were demonstrated. Similar determinations in C.B-20 mice, Ighb congenics of the BALB/c strain, and in C57BL/10 nude mice indicated that the mitogen-generated paratope frequencies directly reflected the capacity of immunoglobulin variable region elements rather than complex interactive or regulatory controls to generate diversity. We conclude that at least for the paratopic repertoire, the role of the somatic processes for the generation of antibody diversity exceeds the influence of germ-line differences between the Ighb and Igha haplotypes.     

Genetics of the anti-dextran B512 and the autoanti-idiotypic response: Codominant expression in F1 hybrids and dichotomy of response and allotype-linked idiotype

The IgM plaque-forming response to the alpha 1–6 epitope of dextran B512 is linked to the Ig-1 heavy chain allotypes j and b characteristic of CBA and C57BL strains, respectively, and the response typically induces the formation of autoanti-idiotypic antibodies that can distinguish between anti-dextran antibodies of CBA and C57BL origin. Nevertheless, some substrains of Balb/c mice (allotype a) and some Bailey recombinant stains give a PFC response although they do not possess allotypes j or b. The anti-dextran antibodies in these strains lack the idiotypes characteristic of either CBA and C57BL antibodies to dextran, but they possess their own particular idiotype. F₁ hybrids between two responder strains possessing different idiotypes on their antibodies against dextran, produce both idiotypes and two different autoanti-idiotypic antibodies. CBA(Ig-1^b) mice were high responders to dextran and possessed the idiotype of C57BL, whereas C57BL/6(Ig-1^a) mice were low responders. The V_H recombinant strains BAB.14 and CB-8KN that possess the Ig-1^b allotype of C57BL, but have some of theV _H genes from Balb/c and the rest from C57BL/6 were high responders to dextran, but did not possess the C57BL idiotype, suggesting that the genes determining the response against dextran and the idiotype may have different locations in the heavy chain locus.     

BALB/c antiarsonate idiotypes: Gene complementation necessary for expression

The expression of two idiotype (id) families (5AF6 and 3C6) associated with the BALB/c p-azophenylarsonate-specific antibody response was examined in 11 mouse strains. Eight strains produced some of one or the other of these two id families with the mean percent expression in the anti-Ar responses of id(+) strains ranging from 8 to 43% for the 5AF6 and from 2 to 10% for the 3C6 idiotype. Four strains of mice (C58, AKR, PL, and RF) thought to have Lyt-3.1-linked V_L repertoire differences from other mouse strains (Lyt-3.2) were tested for their capacity to contribute to 5AF6 and 3C6 id expression. The RE strain was capable of producing 5AF6 id and small amounts of 3C6 id. Tests of Lyt-3.1 congenic strains C.AKR (AKR Lyt-3.1 on a BALB/c background) and C.C58 (C58 Lyt-3.1 on a BALB/c background) showed that C.AKR could produce 5AF6 id while C.C58 could not. 3C6 id expression was present but depressed in C.C58 mice compared with the high 3C6 id expression in C.AKR. Breeding studies mating C.C58 (bearing the required Igh-C^a-linked V _H genes) to other 5AF6(–) strains showed that gene complementation could result in 5AF6 expression in F₁ offspring. 5AF6(–) strains capable of complementation included CBA/J, C57BL/6J, AKR/J, and PL/J. C58/J (from which C.C58 were derived) was the only tested strain that failed to complement for 5AF6 id expression. Additional matings between C58/J[5AF6(–)] and CBA/J[5AF6 (–)] showed F1 offspring could produce 5AF6 id, indicating that C58/J can contribute functional V _H genes necessary for 5AF6 id expression. Depressed expression of 5AF6 and 3C6 id was noted in mice where the C58/J-derived Lyt-3.1 genotype was present. The possibility that the depression of 5AF6 and 3C6 id expression derived from C58/J mice was due to regulatory influences rather than a lack of the V _L structural genes is discussed.Abbreviations used in this paper Ar p-azophenylarsonate - DNP dinitrophenol - H heavy - HIS hyperimmune suppressed - id idiotype - Ig immunoglobulin - IEF isoelectric focusing - k kappa - L light - PC phosphorylcholine - S.D. standard deviation     

Fine-specificity of the contact sensitivity to 4-hydroxy-3-nitrophenyl acetyl (NP)

Dorf and colleagues (1-4) found that the contact sensitivity (CS) primed with (4-hydroxy-3-nitrophenyl)acetyl (NP) could be elicited as easily with the iodoanalog (NIP) as with NP when studied in Igh-1b mice but could only be elicited with NP, not NIP, in Igh-1j mice. Since this fine-specificity was parallel to the fine-specificity of anti-NP antibodies in the two types of mice and since anti-NP antibodies of Igh-1b mice are controlled by gene Igh-NPb the authors concluded that CS also was controlled by the Igh-NPb gene. The aim of this study was to confirm their findings with a more quantitative method (5). We confirmed equality of NP and NIP as elicitors of NP-primed CS in Igh-1b mice when the priming antigen was given subcutaneously into non-cyclophosphamide-treated mice (their method). We also found that this priming induced an anti-NP antibody response detectable at the time of challenge. Most experiments were carried out with a method that does not induce a detectable antibody response (pretreatment of mice with 200 mg/kg of cyclophosphamide; application of the sensitizing compound on skin). Since the NP-primed (and NBrP-primed) CS reactions exhibited "expected specificities," the immunizing compound was clearly the most efficient elicitor (relative efficiencies of homologs varied from 2 to 4). The Igh-NPb gene appears not to have a role in "antibody-free" reactions.     

Genetic controls of T cell-independent Thy-1 alloantibody responses

Early and late primary IgM antibody responses of mice to Thy-1.1 antigens showed different antigenic and cellular requirements. We studied genetic controls of the early primary responses, which could be induced by subcellular thymocyte antigens independently of host T-cell activity. All Thy-1.2 mouse strains of Igh ^a(BALB/c and BC8), Igh-V ^aC^b(BAB14), Igh ^d(AKR/Cum), Igh ^j(CBA/J, C3H/HeN, C3H.SW, and C3H.JK), and Igh ⁿ(NZB) definitely responded early to Thy-1.1 antigens from AKR/J (Igh ^d), A.Thy-1.1 (Igh ^e), or B10.Thy-1.1 (Igh ^b) mice or SD rats, whereas all strains of Igh ^b(C57BL/6, C57BL/10, B10.D2, B10.BR, B10.A, CB20 and CWB), Igh ^c(DBA/2), Igh ^e(A/J), and Igh ^o(C.AL20) responded poorly to the same antigens. This contrasts with the observation that both strains of Igh ^j(C3H/HeN) and Igh ^b(B10.BR) responded well at later times. As was the case for late responses, the matching of H-2 between donor and recipient resulted in early responses of exceptional quality in high-responder strains. It was concluded that under the influence of H-2, whose incompatibility between donor and recipient partially interferes with responses, early but not late primary Thy-1.1-specific antibody responses are selectively controlled by Igh-V or closely linked Ir gene(s) as a new V _Hmarker.Abbreviations used in this paper Tl T cell-independent - TD T cell-dependent - PFC plaque-forming cell(s) - Igh immunoglobulin heavy chain - V _H variable region of heavy chain - C _H constant region of heavy chain     

DonorIgh-linked genetic control of allotype-specific antibody response

Immunogenicity of allogeneic immunoglobulins in mice were studied, measuring the allotype-specific antibody activity by agglutination of allogeneic antibody-coated red blood cells. It was found that the serum from C.B-20 mice (Igh ^b , BALB/c-congenic) was uniquely immunogenic in BALB/c mice for allotype antibody response. Whereas the C57BL/6 (Igh ^b ) serum was immunogenic only when heat aggregated and/or combined with adjuvant, the ultracentrifugation-deaggregated C.B-20 serum was definitely immunogenic when administered in a moderate dose (100 μl/mouse). Even more surprising was the fast that very low doses (0.01–0.1 μl) of soluble C.B-20 serum, but not C57BL/6 serum, down regulated the allotype-specific response effectively. Genetic analysis on congenic mice suggested that the immunogenicity is controlled by donorIgh orIgh-V(Id-C.B) inasmuch as the serum from BALB/c-congenic C.B-20 (Igh-V ^b C ^b ), but not BALB/c-congenic BAB/14 (Igh-V ^a C ^b ), mice was active in BALB/c mice in soluble form. Further studies showed that the Id-C.B was dominantly expressed on the immunoglobulins of (BALB/c×C.B-20)F₁ and (C56BL/6×C.B-20)F₁ strains, and was originally derived from the C57BL/Ka strain. The major determinant for the antibody production was encoded inIgh-C, but not inIgh-V. It is suggested thatId-C.B controls the allotype-specific antibody response in an unusual manner, possibly acting as a unique determinant activating helper T cells.     

Murine responses to (Tyr-Glu-Ala-Gly)n: II. H-2 and non-H-2 gene effects

Mice of the H-2^b haplotype responded to the sequential polymer poly(Tyr-Glu-Ala-Gly) in the in vitro T-cell proliferative assay, irrespective of whether they were homozygous or heterozygous at the H-2^b locus. The antibody responses of the H-2^b congenic mice to this polymer were variable, with A.BY and BALB.B showing responses better than those of C57BL/6 and C57BL/10 strains. The antibody responses of the F₁ progeny of (responder × nonresponder) strains of mice to this polymer are generally lower than the responder parents. F₁ mice with C57BL/10 background were the poorest responders. Studies with F₂ mice and backcross progenies of selective breeding of high and low antibody responder (C57BL/6 × BALB/c) F₁ to high responder C57BL/6 mice indicated that both non-H-2 genes and H-2 gene dosage effects influenced the magnitude of the humoral antibody responses. Animals having low responder non-H-2 background and only half the dosage of the responder immune response genes has greatly diminished antibody responses.     

Hepatic Canalicular Membrane Transport of Bile Salt in C57L/J and AKR/J Mice: Implications for Cholesterol Gallstone Formation

C57L/J (gallstone-susceptible) and AKR/J (gallstone-resistant) mice have been utilized for quantitative trait loci (QTL) analysis to identify the Lith 1 locus for cholelithiasis. Abcb11 encodes for the liver canalicular membrane bile salt export pump (BSEP), which maps to this QTL and is a candidate gene for Lith 1. We investigated the transmembrane transport of taurocholate in canalicular liver membrane vesicles isolated from these murine strains. Canalicular liver plasma membranes (cLPM) and RNA were isolated from C57L/J and AKR/J mice livers, and were utilized for Northern and Western blot analysis and functional ³H-taurocholate uptake studies. ATP-dependent ³H-taurocholate uptake was significantly higher in AKR/J, compared to C57L/J mice. V _max was 127 vs. 42 pmol TC/mg/s in the murine strains, respectively, while K _m was unchanged. In contrast, gene and protein expression of hepatic Abcb11 was increased three-fold in C57L/J, compared to AKR/J mice. Thus, Abcb11 bile salt transport activity per unit protein was reduced nine-fold in the C57L/J, compared to AKR/J mice. In contrast, canalicular membrane cholesterol:phospholipid content was also significantly higher in the C57L/J mice. We conclude that gallstone-susceptible C57L/J mice demonstrate increased gene and canalicular membrane expression of Abcb11, however, taurocholate transport is functionally diminished. The latter may be due to the increased cholesterol membrane content of the cLPM in C57L/J mice. These findings may be important for the pathogenesis of gallstone formation.     

Noninvasive Evaluation of Metabolic Tumor Volume in Lewis Lung Carcinoma Tumor-Bearing C57BL/6 Mice with Micro-PET and the Radiotracers 18F-Alfatide and 18F-FDG: A Comparative Analysis

Purpose

To explore the value of a new simple lyophilized kit for labeling PRGD₂ peptide (¹⁸F-ALF-NOTA-PRGD₂, denoted as ¹⁸F-alfatide) in the determination of metabolic tumor volume (MTV) with micro-PET in lewis lung carcinoma (LLC) tumor-bearing C57BL/6 mice verified by pathologic examination and compared with those using ¹⁸F-fluorodeoxyglucose (FDG) PET.

Methods

All LLC tumor-bearing C57BL/6 mice underwent two attenuation-corrected whole-body micro-PET scans with the radiotracers ¹⁸F-alfatide and ¹⁸F-FDG within two days. ¹⁸F-alfatide metabolic tumor volume (V_RGD) and ¹⁸F-FDG metabolic tumor volume (V_FDG) were manually delineated slice by slice on PET images. Pathologic tumor volume (V_Path) was measured in vitro after the xenografts were removed.

Results

A total of 37 mice with NSCLC xenografts were enrolled and 33 of them underwent ¹⁸F-alfatide PET, and 35 of them underwent ¹⁸F-FDG PET and all underwent pathological examination. The mean ± standard deviation of V_Path, V_RGD, and V_FDG were 0.59±0.32 cm³ (range,0.13~1.64 cm³), 0.61±0.37 cm³ (range,0.15~1.86 cm³), and 1.24±0.53 cm³ (range,0.17~2.20 cm³), respectively. V_Path vs. V_RGD, V_Path vs. V_FDG, and V_RGD vs. V_FDG comparisons were t = -0.145, P = 0.885, t = -6.239, P<0.001, and t = -5.661, P<0.001, respectively. No significant difference was found between V_Path and V_RGD. V_FDG was much larger than V_RGD and V_Path. V_RGD seemed more approximate to the pathologic gross tumor volume. Furthermore, V_Path was more strongly correlated with V_RGD (R = 0.964,P<0.001) than with V_FDG (R = 0.584,P<0.001).

Conclusions

¹⁸F-alfatide PET provided a better estimation of gross tumor volume than ¹⁸F-FDG PET in LLC tumor-bearing C57BL/6 mice.     

Heterologous expression and biochemical characterization of acetyl xylan esterase from Coprinopsis cinerea

Acetyl xylan esterase (AXE) from basidiomycete Coprinopsis cinerea Okayama 7 (#130) was functionally expressed in Pichia pastoris with a C-terminal tag under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 1.5 mg l^?1. Its molecular mass was estimated to be 65.5 kDa based on the SDS-PAGE analysis, which is higher than the calculated molecular mass of 40 kDa based on amino acid composition. In-silico analysis of the amino acid sequence predicted two potential N-glycosylation sites. Results from PNGase F deglycosylation and mass spectrum confirmed the presence of N-glycosylation on the recombinant AXE with predominant N-glycans HexNAc₂Hex_9–16. The recombinant AXE showed best activity at 40 °C and pH 8. It showed not only acetyl esterase activity with a K_m of 4.3 mM and a V_max of 2.15 U mg^?1 for hydrolysis of 4-nitrophenyl acetate but also a butyl esterase activity for hydrolysis of 4-nitrophenyl butyrate with a K_m of 0.11 mM and V_max of 0.78 U mg^?1. The presence of two additional amino acid residues at its native N-terminus was found to help stabilize the enzyme against the protease cleavages without affecting its activity.     

Genetic control of IgM responses to (T,G)-A — L

The primary antibody response to aqueous immunization with a low molecular-weight lot of (T,G)-A — L (#420) was studied in congenic pairs of inbred mouse strains. Two new genetic controls were identified, both of which quantitatively regulate the production of IgM anti-(T,G)-A — L antibody. Testing of F₁ and F₂ progeny demonstrated that one of these genes is linked to the major histocompatibility (H-2) complex, and that high response is dominant over low response. Whether this gene is identical toIr-1A is not yet known. The other gene, designatedIg-TGAL _M , is linked to the immunoglobulin heavy-chain allotype locus (Ig-1) and is expressed in a genedose dependent manner. Following secondary challenge with (T,G)-A — L 420, quantitative differences in IgG antibody response were observed inIr-1A high-responder congenics differing only at theIg-1 locus. Breeding studies, however, failed to demonstrate any linkage between this locus and the quantitative control of IgG anti-(T,G)-A — L antibody. These data demonstrate thatH-2-linked immune response genes can regulate IgM as well as IgG antibody responses, that genetic control of the IgM response to (T,G)-A — L is linked toIg-1, and that bothH-2-linked andIg-1-linked genes may simultaneously affect an IgM antibody response to the same antigen.Abbreviations used in this paper are (T,G)-A — L poly-l-(Tyr,Glu)-poly-d,l-Ala-poly-l-Lys - NMS normal mouse serum - SRBC sheep red blood cells - i.p. intraperitoneal - PBS phosphate-buffered saline - RAMG polyvalent rabbit anti-mouse globulin - 2-Me 2-Mercaptoethanol - 2-MeS 2-Me-sensitive - PFC plaque-forming cells - ABC antigen-binding cells     

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

T-cell receptor-specific monoclonal antibodies against a V β 11-positive mouse T-cell clone

Two monoclonal antibodies specific for the mouse T-cell receptor (Tcr) have been established by immunization with a V 11⁺ T-cell clone, clone C6. One is a rat antibody, KT11 (IgG2b, k), specific for the V chain of C6, V 11. This was demonstrated by the fact that the strain distribution pattern of KT11⁺ cells was similar to that of V 5, 8, 9, 11, 12, and 13 and that the gene that encodes the molecule detected by KT11 was closely linked to V 8 in (B10 × SJL)F₁ × SJL backcross mice. Furthermore, V of C6 has been cloned from a gt10 cDNA library and was demonstrated to be identical to the V 11 published sequences. All strains of mice that do not express major histocompatibility complex class II E molecules had higher numbers of KT11^– cells than E⁺ strains. The KT11⁺ population in A strain mice and its H-2 congenic strains, however, was not affected by the presence or absence of E molecules. The other is a mouse antibody, KTL2 (IgM), specific for the idiotope of the Tcr expressed on the clone C6. Both antibodies were mitogenic and induced cytotoxicity. Expression of epitopes detected by KT11 or KTL2 was down-modulated by a T3-specific antibody 145-2C11.     

Purification and Characterization of an Esterase Involved in Poly(vinyl alcohol) Degradation by Pseudomonas vesicularis PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45°C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K _m and V _max of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 μM, and 6.52 and 12.6 μmol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.     

A Catalytic Antibody That Accelerates the Hydrolysis of Carbonate Esters. Prediction of the Binding-Site Structure of the Substrate

Monoclonal antibodies catalyzing the hydrolysis of p-nitrophenyl alkyl carbonate were obtained using p-nitrophenyl phosphonate as hapten. One of the antibodies, 4A1, has a relatively high activity for the substrate having a bulky group. To determine the amino acid residues related to the binding of the bulky group, we determined the amino acid sequences of V_L and V_H regions of 4A1 by the cycle sequencing method, built the three-dimensional structure of the V regions, labeled 4A1 with [¹⁴C]phenyl glyoxal in the presence and absence of I-1 or I-13, and analyzed the labeled incubation mixture with SDS–PAGE. From these results, the possibility that Arg-H28 of the heavy chain is involved in binding the bulky group of the substrate is discussed.     

Adenosine A2A receptor and vascular response: role of soluble epoxide hydrolase,adenosine A1 receptor and angiotensin-II

Previously, we have reported that the coronary reactive hyperemic response was reduced in adenosine A_2A receptor-null (A_2AAR^?/?) mice, and it was reversed by the soluble epoxide hydrolase (sEH) inhibitor. However, it is unknown in aortic vascular response, therefore, we hypothesized that A_2AAR-gene deletion in mice (A_2AAR^?/?) affects adenosine-induced vascular response by increase in sEH and adenosine A₁ receptor (A₁AR) activities. A_2AAR^?/? mice showed an increase in sEH, A_I AR and CYP450-4A protein expression but decrease in CYP450-2C compared to C57Bl/6 mice. NECA (adenosine-analog) and CCPA (adenosine A₁ receptor-agonist)-induced dose-dependent vascular response was tested with t-AUCB (sEH-inhibitor) and angiotensin-II (Ang-II) in A_2AAR^?/? vs. C57Bl/6 mice. In A_2AAR^?/?, NECA and CCPA-induced increase in dose-dependent vasoconstriction compared to C57Bl/6 mice. However, NECA and CCPA-induced dose-dependent vascular contraction in A_2AAR^?/? was reduced by t-AUCB with NECA. Similarly, dose-dependent vascular contraction in A_2AAR^?/? was reduced by t-AUCB with CCPA. In addition, Ang-II enhanced NECA and CCPA-induced dose-dependent vascular contraction in A_2AAR^?/? with NECA. Similarly, the dose-dependent vascular contraction in A_2AAR^?/? was also enhanced by Ang-II with CCPA. Further, t-AUCB reduced Ang-II-enhanced NECA and CCPA-induced dose-dependent vascular contraction in A_2AAR^?/? mice. Our data suggest that the dose-dependent vascular contraction in A_2AAR^?/? mice depends on increase in sEH, A₁AR and CYP4A protein expression.

     

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

Genetic control of the target structures recognized in hybrid resistance

Hybrid resistance of lethally irradiated (C57BL/6 × DBA/2)F₁ and (C57BL/10 × C3H)F₁ hybrid mice to the engraftment of parental C57BL/6 or C57BL/10 bone marrow cells is controlled by the H-2-linked Hh-1 locus. This resistance can be specifically blocked or inhibited by the injection of irradiated spleen cells from lethally irradiated, marrow reconstituted donor mice of certain strains. By testing the ability of regenerating spleen cells from various donor strains to block the resistance, we studied the genetic requirements for the expression of putative cell-surface structures recognized in hybrid resistance to H-2^b marrow cells. Strains of mice bearing informative intra-H-2 or H-2/ Qa-Tla recombinant haplotypes provided evidence that the Hh-1 locus is located telomeric to the H-2S region complement loci and centromeric to the H-2D region class I locus in the H-2 ^b chromosome. Two mutations that affect the class I H-2D ^b gene have no effect on Hh-1 ^b gene expression. The H-2D region of the H-2 ^S haplotype contains an allele of the Hh-1 locus indistinguishable from that of the H-2D ^b region, as judged by the phenotypes of relevant strains and F₁ hybrids. Collectively these data indicate that the Hh-1 locus is distinct from the class I H-2D (L) locus in the H-2 ^b or H-2 ^s genome, and favor the view that the expression or recognition of the relevant determinants is not associated with class I gene products.Abbreviations used in this paper BM(C) bone marrow (cells) - CML cell-mediated lympholysis - CTL cytotoxic T lymphocytes - FBS fetal bovine serum - HBSS Hanks' balanced salt solution - SC spleen cells from irradiated, bone marrow-reconstituted mice Address correspondence to: Dr. I. Najamura, Department of Pathology, School of Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA