Trihydroxytetraenes: A novel series of compounds formed from arachidonic acid in human leukocytes

Addition of 15L-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) to human leukocytes led to the formation of a novel series of compounds containing four conjugated double bonds. The yield of tetraenes was increased approx. 100-fold when ionophore A23187 (5 μM) was added simultaneously with 15-HPETE. The structure of the major tetraene was established by physical methods as well as by chemical degradation and found to be 5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid.     

重组大肠杆菌YK537/pSB┐TK高密度培养过程中有机酸的RP┐HPLC快速分析

采用高压液相色谱法（ＨＰＬＣ）,利用Ｃ１８反相柱快速检测重组大肠杆菌ＹＫ５３７／ｐＳＢ－ＴＫ高密度发酵过程中细菌代谢产生的各种有机酸。分析表明,经乙醚和水两次萃取处理后能去除样品中大部分的干扰峰,可获得理想的分离效果,并能定量测出发酵过程中各种有机酸的积累。不同培养方式的结果表明用甘油代替葡萄糖作为细菌生长的碳源可减少乙酸的积累,降低其对细菌生长和外源蛋白表达的抑制作用,从而更易实现重组大肠杆菌的高表达、高密度培养。     

Structural determinants of the selectivity of KTS-disintegrins for the alpha1beta1 integrin

KTS-disintegrins are a subfamily of short monomeric disintegrins that are potent and selective inhibitors of alpha1beta1 integrin. The amino acid sequence of the new KTS-disintegrin, viperistatin, differs from previously characterized obtustatin in three residues at position 24 (within the integrin binding loop), 38 (hydrophobic core) and 40 (C-terminal region). Noteworthy, viperistatin is about 25-fold more potent than obtustatin inhibiting the binding of this integrin to collagen IV. Synthetic peptides representing the full-length of integrin-binding loops of these disintegrins showed that the Leu24/Arg substitution appears to be partly responsible for the increased inhibitory activity of viperistatin over obtustatin.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Characteristics of the epitope of leukotriene B4 recognized by a highly specific mouse monoclonal antibody

Mouse monoclonal IgG2b antibodies to leukotriene B4 bind [3H]leukotriene B4 with an affinity one-thirtieth to one-third that of different rabbit antibodies to leukotriene B4. The concentrations of related ligands required to inhibit by 50% the binding of [3H]leukotriene B4 define cross-reactivities of approximately 100% for carboxyl-derivatives of leukotriene B4, 10% for 12(S)-leukotriene B4 and 8 cis-leukotriene B4, which were not distinguished from leukotriene B4 by polyclonal antibodies, 3-5% for the two isomers of 6 trans-leukotriene B4, 5% for 20-OH-leukotriene B4 and 20-COOH-leukotriene B4, and less than 1% for other leukotrienes, mono-hydroxy-eicosatetraenoic acids, and the two leukotriene B4-like isomers of 8, 15-di-hydroxy-eicosatetraenoic acid. Thus the monoclonal combining site is highly specific for the di-hydroxy-triene portion of leukotriene B4.     

Group X secretory phospholipase A2 induces potent productions of various lipid mediators in mouse peritoneal macrophages

We have previously shown the expression of group X secretory phospholipase A₂ (sPLA₂-X) in mouse splenic macrophages and its powerful potency for releasing fatty acids from various intact cell membranes. Here, we examined the potency of sPLA₂-X in the production of lipid mediators in murine peritoneal macrophages. Mouse sPLA₂-X was found to induce a marked release of fatty acids including arachidonic acid and linoleic acid, which contrasted with little, if any, release by the action of group IB and IIA sPLA₂s. In resting macrophages, sPLA₂-X elicited a modest production of prostaglandin E₂ and thromboxane A₂. After the induction of cyclooxygenase-2 (COX-2) by pretreatment with lipopolysaccharide, a dramatic increase in the production of these eicosanoids was observed in sPLA₂-X-treated macrophages, which was completely blocked by the addition of either the specific sPLA₂ inhibitor indoxam or the COX inhibitor indomethacin. In accordance with its higher hydrolyzing activity toward phosphatidylcholine, mouse sPLA₂-X induced a potent production of lysophosphatidylcholine. These findings strongly suggest that sPLA₂-X plays a critical role in the production of various lipid mediators from macrophages. These events might be relevant to the progression of various pathological states, including chronic inflammation and atherosclerosis.     

The mitogenic activity of insulin: An intrinsic property of the molecule

Higher than physiological concentrations of insulin stimulate the incorporation of [${}^3\text{H}$]thymidine into DNA in a large variety of cells in culture. In order to exclude a contaminant of insulin preparations as responsible for this mitogenic action, porcine insulin was purified by reverse phase HPLC and assayed for mitogenic activity. HPLC- purified insulin had a mitogenic activity similar to that of impure insulin. Moreover, semisynthetic and synthetic insulins as well as HPLC-purified insulin derivatives such as monodesamido- and monoarginine insulin were also biologically active.     

A central role for intermolecular dityrosine cross-linking of fibrinogen in high molecular weight advanced oxidation protein product (AOPP) formation

Background

Advanced oxidation protein products (AOPPs) are dityrosine cross-linked and carbonyl-containing protein products formed by the reaction of plasma proteins with chlorinated oxidants, such as hypochlorous acid (HOCl). Most studies consider human serum albumin (HSA) as the main protein responsible for AOPP formation, although the molecular composition of AOPPs has not yet been elucidated. Here, we investigated the relative contribution of HSA and fibrinogen to generation of AOPPs.

Methods

AOPP formation was explored by SDS-PAGE, under both reducing and non-reducing conditions, as well as by analytical gel filtration HPLC coupled to fluorescence detection to determine dityrosine and pentosidine formation.

Results

Following exposure to different concentrations of HOCl, HSA resulted to be carbonylated but did not form dityrosine cross-linked high molecular weight aggregates. Differently, incubation of fibrinogen or HSA/fibrinogen mixtures with HOCl at concentrations higher than 150 μM induced the formation of pentosidine and high molecular weight (HMW)-AOPPs (> 200 kDa), resulting from intermolecular dityrosine cross-linking. Dityrosine fluorescence increased in parallel with increasing HMW-AOPP formation and increasing fibrinogen concentration in HSA/fibrinogen mixtures exposed to HOCl. This conclusion is corroborated by experiments where dityrosine fluorescence was measured in HOCl-treated human plasma samples containing physiological or supra-physiological fibrinogen concentrations or selectively depleted of fibrinogen, which highlighted that fibrinogen is responsible for the highest fluorescence from dityrosine.

Conclusions

A central role for intermolecular dityrosine cross-linking of fibrinogen in HMW-AOPP formation is shown.

General significance

These results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.     

Correlation between the phospholipids domains of the target cell membrane and the extent of Naja kaouthia PLA2-induced membrane damage: Evidence of distinct catalytic and cytotoxic sites in PLA2 molecules

Two phospholipase A₂ (PLA₂) enzymes (NK-PLA₂-A and NK-PLA₂-B) were purified from the venom of the monocled cobra Naja kaouthia. The molecular weights of NK-PLA₂-A and NK-PLA₂-B, as estimated by mass spectrometry, were 13,619 and 13,303 Da respectively. Both phospholipases were highly thermostable, had maximum catalytic activity at basic pH, and showed preferential hydrolysis of phosphatidylcholine. Intravenous injection of either PLA₂ up to a dose of 10 mg/kg body weight was non-toxic to mice and did not show neurotoxic symptoms. The N. kaouthia PLA₂s displayed anticoagulant and cytotoxic activity, but poor hemolytic activity. Both the PLA₂s were more toxic to Sf9 and Tn cells compared to VERO cells. NK-PLA₂ exhibited selective lysis of wild-type baculovirus-infected Sf9 cells compared to normal cells. Amino acid modification studies and heating experiments suggest that separate sites in the NK-PLA₂ molecules are responsible for their catalytic, anticoagulant and cytotoxic activities.     

Isolation of the first toxin from the scorpion Buthus occitanus israelis showing preference for Shaker potassium channels

We have purified BoiTx1, the first toxin from the venom of the Israeli scorpion, Buthus occitanus israelis, and studied its activity and genomic organization. BoiTx1 is a 37 amino acid-long peptide contained six conserved cysteines, and is classified as an alpha-KTx3.10 toxin. The pharmacological effects of BoiTx1 were studied on various cloned K(+) channels expressed in Xenopus laevis oocytes. BoiTx1 inhibited currents through Drosophila Shaker channels with an IC(50) value of 3.5+/-0.5nM, yet had much lesser effect on its mammalian orthologs. Thus, BoiTx1 is the first member of the alpha-KTx3 family that preferentially affects insect potassium channels.     

Neuromedin B is a major bombesin-like peptide in rat brain: regional distribution of neuromedin B and neuromedin C in rat brain, pituitary and spinal cord

Neuromedin B and neuromedin C are the novel mammalian bombesin-like peptides isolated from porcine spinal cord. We have developed highly specific and sensitive radioimmunoassays for neuromedin B and neuromedin C, and determined their regional distribution in rat central nervous system. Prior to measurements of the tissue contents, immunoreactive neuromedin B and C were characterized by gel-filtration and high performance liquid chromatography. Neuromedin B and C immunoreactivities have similar regional distribution in rat brain, but the content of immunoreactive neuromedin B is 2-6 times higher than that of immunoreactive neuromedin C in every region. These results indicate that neuromedin B is a major endogenous bombesin-like peptide in rat brain and has specific functions of physiological importance.     

Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.     

Regional distribution of neuromedin K and neuromedin L in rat brain and spinal cord

Neuromedin K and neuromedin L are novel mammalian tachykinins isolated from porcine spinal cord. We have developed a highly sensitive radioimmunoassay for neuromedin K. Since the radioimmunoassay for neuromedin K has significant crossreactivity with neuromedin L and substance P, we can simultaneously determine the tissue concentrations of neuromedin K, neuromedin L and substance P after separation of the tissue extracts by reverse phase high performance liquid chromatography. Substance P is found to be the most abundant mammalian tachykinin in every brain region. The ratio of the concentration of substance P to neuromedin K is small in cerebral cortex and large in medulla-pons, while that of substance P to neuromedin L is rather constant in a range of 2.0–2.5. In spinal cord, dorsal half contains more neuromedin K and L than ventral half as is the case with substance P. These results indicate that both neuromedin K and L are endogenous mammalian tachykinins with specific physiological functions.     

Identification of interleukin-8 converting enzyme as cathepsin L

IL-8 is produced by various cells, and the NH₂-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH₂-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH₂-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg⁵ and Ser⁶, which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.     

Use of vitamin D4 analogs to investigate differences in hepatic and target cell metabolism of vitamins D2 and D3

In this study, we used molecules with either of the structural differences in the side chains of vitamin D₂ and vitamin D₃ to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems—HepG2 and HPK1A-ras—to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1α-hydroxyvitamin D₂ (1α-OH-D₂) but not for 1α-OH-D₃ is also observed in both 1α-OH-D₄ and Δ²²-1α-OH-D₃ metabolism. This suggests that the structural feature responsible for targeting the enzyme to the C24 or C26 site could be either the C24 methyl group or the 22–23 double bond. In HPK1A-ras cells, the pattern of metabolism observed for the 24-methylated derivative, 1α,25-(OH)₂D₄, was the same pattern of multiple hydroxylations at C24, C26 and C28 seen for vitamin D₂ compounds without evidence of side chain cleavage observed for vitamin D₃ derivatives, suggesting that the C24 methyl group plays a major role in this difference in target cell metabolism of D₂ and D₃ compounds. Novel vitamin D₄ compounds were tested and found to be active in a variety of in vitro biological assays. We conclude that vitamin D₄ analogs and their metabolites offer valuable insights into vitamin D analog design, metabolic enzymes and maybe useful clinically.     

Molecular heterogeneity of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes

The molecular heterogeneity of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes (PMN) was assessed by both normal and reverse phase high performance liquid chromatography (HPLC). As detected by rabbit platelet stimulation, at least 5 PAF molecules were separated by HPLC. Fast atom bombardment (FAB) mass spectrometry revealed one of these PAFs was acetyl glyceryl ether phosphorylcholine (AGEPC) with a C_16:0 alkyl chain in the $\text{sn}$-1 position. Although the structures of the remaining PAFs are unknown, two of the peaks of PAF activity had the same retention times on reverse phase HPLC as the C₁₅- and C₁₈-saturated alkyl chain AGEPC homologues. These studies indicate that the human PMN produces multiple molecular species of PAF.     

Gelatin binding to the 8F19F1 module pair of human fibronectin requires site-specific N-glycosylation

The gelatin (denatured collagen) binding domain of the extracellular matrix protein fibronectin contains three potential N-glycosylation sites. Complete deglycosylation of this domain is known to reduce the thermal stability of the eighth type 1 (8F1) module. We have conducted a site-specific analysis of the structural and functional consequences of N-linked glycosylation in the 8F19F1 module pair. Three glycoforms have been identified by mass spectrometry and nuclear magnetic resonance spectroscopy. Chemical shift differences between the glycoforms have revealed an intimate interaction between one N-linked sugar and the polypeptide that is critical for gelatin binding, as shown by affinity chromatography.