Hepatic drug-metabolizing enzyme system and endotoxin tolerance: structural requirement of LPS in induction of an early tolerance

The alteration of hepatic drug-metabolizing enzyme activities in mice given Salmonella endotoxin by single or multiple intraperitoneal injections was investigated. An essentially the same biphasic, early and late phase, endotoxin tolerance was observed in the animals receiving a single injection of endotoxin or repetitive daily injections. The results of reciprocal cross tolerance tests using lipopolysaccharide and free lipid A preparations derived from Salmonella minnesota, Salmonella typhimurium, E. coli, Pseudomonas aeruginosa, and Chromobacterium violaceum suggested that lipid A moiety plays an important role in the induction of early endotoxin tolerance to endotoxin response.     

Electric charge distribution in proteins and ionic strength dependence of reaction rate

The ionic strength dependence of the reaction rate between protein and dichloride anion radical has been investigated by flash photolysis of aqueous chloride-containing lysozyme, ribonuclease A, or insulin. The rate constant for the reaction of lysozyme or ribonuclease A with dichloride anion radicals decreases with increasing ionic strength, while it increases for insulin. The dependence was found to obey an equation derived from the theory of Debye and Hückel or the equation of Wherland and Gray for lysozyme within experimental errors. For ribonuclease A, however, it deviates largely from these equations. In the case of insulin a moderate deviation was observed. The different behavior in the ionic strength dependence is discussed in terms of the electric charge distribution in the protein molecules.     

Structural heterogeneity regarding local Shwartzman activity of lipid A

The relation of chemical structure to local Shwartzman activity of lipid A preparations purified by thin-layer chromatography from five bacterial strains was examined. Two lipid A fractions from E. coli F515--Ec-A2 and Ec-A3--exhibited strong activity, similar to that of previous synthetic E. coli-type lipid A (compound 506 or LA-15-PP). The Ec-A3 fraction contained a component that appeared to be structurally identical to compound 506, and the main component of Ec-A2 fraction was structurally similar to compound 506 except that it carried a 3-hydroxytetradecanoyl group at the C-3' position of the backbone in place of a 3-tetradecanoyloxytetradecanoyl group. Free lipid A (12 C) and purified lipid A fractions, Ec-A2 (12 C) and Ec-A3 (12 C), respectively, obtained from bacteria grown at 12 C, exhibited activity comparable to Ec-A2 or Ec-A3. In these preparations, a large part of the 3-dodecanoyloxytetradecanoyl group might be replaced by 3-hexadecenoyloxytetradecanoyl group. Salmonella minnesota R595 free lipid A also contained at least two active lipid A components as seen in E. coli lipid A, but the third component corresponding to the synthetic Salmonella-type lipid A (compound 516 or LA-16-PP) exhibited low activity. A lipid A fraction, Cv-A4 from Chromobacterium violaceum IFO 12614, which was proposed to have two acyloxyacyl groups at the C-2 and C-2' positions with other acyl groups, exhibited weaker activity than the free lipid A or LPS. The purified lipid A fractions from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 contained an unusual backbone with 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester, and these lipid A (Pd-A3 and Pv-A3) exhibited strong activity comparable to the E. coli lipid A. Thus, the present results show that the local Shwartzman reaction can be expressed by partly different lipid A structures in both hydrophilic backbone and fatty acyl residues; when they have the same backbone the potency varies markedly depending on the structure of the acyl residues.     

Characterization of a glucuronyltransferase: neolactotetraosylceramide glucuronyltransferase from rat brain

The properties of a rat brain glucuronyltransferase, which is presumed to be associated with the biosynthesis of the HNK-1 epitope on sulfoglucuronyl glycolipids, are described. The enzyme required divalent cations for reaction, with maximal activity at 10mm Mn²⁺, and exhibited a dual optimum at pH 4–5 and pH 6 depending upon the buffer used, with the highest activity at pH 4.5 in MES buffer. This enzyme strictly recognized the Gal1-4GlcNAc terminal structure, and was highly specific for neolacto (type 2) glycolipids as acceptor. The enzyme was localized specifically in the brain, and was barely detected in other issues, including the thymus, spleen, liver, kidney, lung, and sciatic nerve fibres. Phosphatidylinositol and phosphatidylserine increased the enzymatic reaction 4.4- and 2.3-fold, respectively, whereas phosphatidylcholine slightly decreased the rate.Abbreviations GlcA glucuronic acid - Lc-PA₁₄ lactotetraose-phenyl-C₁₄H₂₉ - nLc-PA₁₄ neolactotetraose-phenyl-C₁₄H₂₉ - nLcOse₄-Cer neolactotetraosylceramide - NP-40 Nonidet P-40 - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SGGL sulfoglucuronyl glycolipid     

Structure of major surface determinants and DNA diagnosis of Pneumocystis carinii

Pneumocystis carinii is a pathogen which causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii, we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called "P115" with apparent masses of 105-120 kd. It includes 6 isoelectric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunoreactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.     

Recognition of the Qa-2k tumor antigen by T cell receptor γ/δ of an immunopotentiator-induced tumoricidal T cell of mice

Tumor-specific expression of Qa-2^k antigen coded by the Q5^k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2^– mice. Lymphocytes stimulated in vivo withP. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (L^Q7b/Kb), which expressed the 1 and 2 domains of the Qa-2 antigen (Q7^b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5 half of the Q7^b gene and the 3 half of the H-2K^b gene (pQ7^b/K^b). Using L^Q7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes fromP. acnes-stimulated (C3H/He × B6.K1)F1 mice (H-2^k, Qa-2^–) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the 1/2 region of the Q7^b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the / type (TCR/). The (C3H/He × B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2^k+) derived from AKR mice (Qa-2^–, H-2^k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed L^Q7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the 1/2 region of the Qa-2^k tumor antigen by TCR/.     

PCR method for genotyping and zygosity-testing of RasH2 transgenic mice.

In short-term carcinogenicity testing using CB6F1-TgrasH2 mice, sibling nonTgrasH2 mice are used as a negative control. However, selection of TgrasH2 and nonTgrasH2 mice has been performed by PCR with only transgene specific primers by the conventional method. Therefore, the conventional method involves the risk of false negative results due to reaction failure, and contamination with TgrasH2 mice in the control mice group. Based on the nucleotide sequence information around the pre-integration site, we developed a genotyping method for distinguishing not only TgrasH2 mice (hemizygous for the Tg allele) but also nonTgrasH2 (homozygous for the nonTg allele) in a positive manner.     

CDP-diglyceride:inositol transferase from rat liver. Purification and properties.

CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.     

Phosphatidyl inositol: myo-Inositol exchange enzyme from rat liver: Partial purification and characterization

The enzyme which catalyzes CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol was solubilized from rat liver microsomes by sodium cholate and was partially purified by ammonium sulfate fractionation and sucrose density gradient centrifugation. Addition of phospholipids during purification and assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation contained about 3.7% of the protein and 35% of the original activity of the microsomal fraction. The activity of the enzyme preparation was strongly enhanced by addition of phosphatidyl inositol. The enzyme required Mn²⁺ for activity. The K_m for myo-inositol was 4 × 10^?5m. The pH optimum was 7.4. The activity was inhibited by thiol-reactive reagents and also to some extent by inosose-2 but not by scyllitol. Phosphorus-containing acidic substances such as acidic phospholipids and nucleotides were generally inhibitory. It was found that the preparation catalyzed liberation of inositol moiety from phosphatidyl inositol in a manner dependent on the concentration of free myo-inositol and also on Mn². The K_m of this reaction for free myo-inositol was estimated to be 7 × 10^?5m. This result indicates that CDP-diglyceride-independent incorporation, which has been assumed to show inositol exchange reaction, actually represents an exchange reaction between the myo-inositol moiety of phosphatidyl inositol and free myo-inositol. Phosphatidyl choline and phosphatidyl ethanolamine did not play a role as acceptor of the exchange reaction.     

Proteins and Carbohydrates in Xylem Sap from Squash Root

The xylem sap from squash roots was collected from the cut surfaceof stems, and the proteins and carbohydrates in the sap wereanalyzed. The sap contained 18.6 µg ml^–1 proteinand the major polypeptides were as follows: 1) two polypeptides,of 75 and 40 kDa, with high-mannose glycans, the levels of whichincreased for about 24 h after cutting and then decreased; 2)a 32-kDa polypeptide, which appeared soon after cutting, disappearedand then reappeared again 48–64 h after cutting; and 3)a 19-kDa and a 14-kDa polypeptide, which were present constitutively.The carbohydrates contained in the xylem sap were fractionatedinto 80% ethanol-soluble and -insoluble material, and whichwere analyzed by high-performance liquid chromatography, gaschromatography and enzymatic mathods. The former fraction containedconsiderable amounts of myo-inositol and fructose as free sugarsand oligosaccharides composed mainly of galactose, arabinoseand glucose. The latter contained polysaccharides composed mainlyof uronic acids, galactose and arabinose. The possible significanceof these substances, which may mediate the interactions betweenthe root and the aerial organs, is discussed. (Received April 20, 1992; Accepted July 4, 1992)