In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as -N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of -N-methyllysines (1.40, 1.66, and 5.62 mol% for -N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of -N-methyllysines in histone H1.     

\4 integrin expression onin vitro andin vivo metastatic variants of lewis lung carcinoma

The expression of 4, 6, and 1 integrin subunits has been investigated on somein vitro andin vivo murine metastatic variants derived from Lewis lung carcinoma (3LL). By the use of monoclonal antibodies which recognizes different epitopes of 6, 1, and 4 subunits we demonstrate that 6 and 1 subunits are expressed in all metastatic variants of 3LL irrespective of their metastatic potential, whereas 4 subunit is expressed only in highly metastasizing cells of 3LL. Northern blots of different metastatic variants probed with 1 and 4 subunits demonstrate thata) significant amounts of 1 mRNA were detected in all metastatic variants of 3LL;b) mRNA corresponding to the described entire coding sequence of 4 subunit is expressed only on highly metastasizing cells of 3LL. We conclude that 4 subunit is specifically expressed in highly metastasizig cells of 3LL while is undetectable in lower metastasizing ones.     

Presence and partial characterisation of a major proteolytic enzyme in the larval gut of Callosobruchus maculatus

Protease activity in the gut of larvae of the bruchid beetle, Callosobruchus maculatus (a storage pest of cowpea seeds), has been investigated to help clarify nutritional mechanisms in view of reports that these insects carry out little or no proteolysis (Applebaum, 1964). Larval gut homogenates showed protease activity against a variety of different protein substrates, but did not hydrolyse a synthetic trypsin substrate. The proteolytic activity had a pH optimum of 5.4. It was not inhibited by serine protease inhibitors, but was inhibited by reagents reactive against-SH groups. Protein trypsin inhibitors from legume seeds which are not hosts to C. maculatus (soybean, limabean) were not effective inhibitors of the larval proteolytic activity but a cowpea protease inhibitor preparation and aprotinin partially inhibited proteolysis. The latter two inhibitors also inhibited the plant thiol protease papain. It is suggested that C. maculatus has replaced the normal insect proteases with an enzyme similar to plant proteases to evade the antimetabolic effects of trypsin/chymotrypsin inhibitors in seeds. Besides trypsin/chymotrypsin inhibitors, cowpea seeds also contain proteins which inhibit papain; these inhibitors were purified and were shown to be effective inhibitors of C. maculatus larval protease.

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Mise en évidence et caractérisation partielle d'une enzyme protéolytique importante du tube digestif des larves de Callosobruchus maculatus

Résumé Callosobruchus maculatus attaque les graines stockées particulièrement de Vigna unguiculata. L'activité protéolytique du tube digestif des larves a été examinée. Aucune hydrolyse n'a été observée contre la N-benzoyl arginine p-nitroanilide (substrat de la trypsine synthétique), mais la protéolyse a été mise en évidence en utilisant la viciline de V. unguiculata comme substrat naturel ou la myoglobine comme substrat artificiel, le p.H optimum est de 5,4. Les inhibiteurs chimiques des protéases de la sérine n'ont pas altéré l'activité enzymatique, mais les réactifs du groupe SH l'ont inhibée. Nous suggérons que cette protéase est une thiol protéase, c'est à dire avec un groupe actif semblable à celui de la papaïne, protéase végétale. En accord avec cette hypothèse, les inhibiteurs de protéine efficaces seulement contre la trypsine, c'est à dire les inhibiteurs de la trypsine de Glycine max et de la trypsine de Phaseolus lunatus n'affectent pas cette protéase larvaire, tandis que les protéines avec une faible action inhibitrice contre la papaïne (préparation inhibitrice de la protéase de V. unguiculata, aprotinine) inhibent partiellement la protéolyse par des extraits de tube digestif larvaire.Des inhibiteurs protéiques de papaïne extraits de graines de V. unguiculata sont des inhibiteurs très efficaces de la protéase larvaire. On peut penser que C. maculatus contient une protéase semblable aux protéases végétales, plutôt qu'aux protéases classiques, d'insectes, ce qui permet d'éviter les effets antimétaboliques directs des inhibiteurs de protéase (inhibiteurs de trypsine et de chymotrypsine) trouvés en quantité relativement importantes dans les graines de légumineuses.

     

The selective inactivation of thiol proteasesin vitro andin vivo

Peptidyl diazomethyl ketone derivatives inactivate thiol proteases by affinity labeling. The peptide sequence permits the reagents to be targeted to individual proteases according to their specificity. Alkylation of the active center thiol group takes place. The reagents are not reactive to simple thiols such as mercaptoethanol and appear to inactivate thiol proteases by acting as suicide substrates. Other classes of proteases are not affected. Initial results indicate that peptidyl diazomethyl ketones can also be used to inactivate thiol proteases in vivo.     

Proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase in Citrus leaf extracts

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) has been purified from orange [ Citrus sinensis (L.) Osbeck cv. Washington Navel] leaves using sucrose gradient centrifugation in a fixed angle rotor. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands corresponding to the two subunits of RuBP carboxylase were found. The large subunit coincided with the polypeptide band that has been previously reported to be preferentially mobilized during the spring and summer flush periods.
The degradation of RuBP carboxylase during autodigestion of Citrus leaf extracts, investigated by SDS-PAGE, occurred mainly at acidic (2.5-5.5) pH. The two subunits showed differences in the rate of degradation, the smaller being more rapidly hydrolyzed than the larger. At least four proteolytic activities were identified by means of inhibitor experiments: 1) a pepstatin A-sensitive activity that acts on both RuBP carboxylase subunits, 2) a mercurial ( p -hydroxymercuribenzoate and p -chloromercuriphenylsulfonate)-sensitive activity that degrades only the small subunit, 3) an EDTA-sensitive activity that hydrolyzes both the large and small subunits, and 4) a mercurial-stimulated activity that acts only on the large subunit. It is suggested that the last two proteases may be responsible for the degradation of RuBP carboxylase observed in vivo during the periods of mobilization of leaf protein in Citrus .     

Novel in vitro and in vivo inhibitors of prolyl endopeptidase

Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.     

金属硫蛋白与红细胞的相互作用

根据金属硫蛋白(MT)对标记在膜上的马来酰亚胺自旋标记物的ESR波谱的影响,研究了MT与红细胞膜的相互作用,发现不同种属的MT对膜构象的影响不同.体外实验表明,MT可以吸附在红细胞的表面,用CdCl₂诱导家兔,对血浆和红细胞溶血液中的MT组分进行色谱分离,发现血液中的MT主要存在于血细胞中.进而对血液MT的来源、分布及其重要的生物学意义进行了讨论.     

Avian 3-hydroxy-3-methylglutaryl-CoA lyase: sensitivity of enzyme activity to thiol/disulfide exchange and identification of proximal reactive cysteines.

Catalysis by purified avian 3-hydroxy-3-methylglutaryl-CoA lyase is critically dependent on the reduction state of the enzyme, with less than 1% of optimal activity being observed with the air-oxidized enzyme. The enzyme is irreversibly inactivated by sulfhydryl-directed reagents with the rate of this inactivation being highly dependent upon the redox state of a critical cysteine. Methylation of reduced avian lyase with 1 mM 4-methylnitrobenzene sulfonate results in rapid inactivation of the enzyme with a k(inact) of 0.178 min-1. The oxidized enzyme is inactivated at a sixfold slower rate (k(inact) = 0.028 min-1). Inactivation of the enzyme with the reactive substrate analog 2-butynoyl-CoA shows a similar dependence upon the enzyme's redox state, with a sevenfold difference in k(inact) observed with oxidized vs. reduced forms of the enzyme. Chemical cross-linking of the reduced enzyme with stoichiometric amounts of the bifunctional reagents 1,3-dibromo-2-propanone (DBP) or N,N'-ortho-phenylene-dimaleimide (PDM) coincides with rapid inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme treated with bifunctional reagent reveals a band of twice the molecular weight of the lyase monomer, indicating that an intersubunit cross-link has been formed. Differential labeling of native and cross-linked protein with [1-14C]iodoacetate has identified as the primary cross-linking target a cysteine within the sequence VSQAACR, which maps at the carboxy-terminus of the cDNA-deduced sequence of the avian enzyme (Mitchell, G.A., et al., 1991, Am. J. Hum. Genet. 49, 101). In contrast, bacterial HMG-CoA lyase, which contains no corresponding cysteine, is not cross-linked by comparable treatment with bifunctional reagent. These results provide evidence for a potential regulatory mechanism for the eukaryotic enzyme via thiol/disulfide exchange and identify a cysteinyl residue with the reactivity and juxtaposition required for participation in disulfide formation.     

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (～99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent K_m value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.     

Phagocytosis by carp granulocytes; in vivo and in vitro observations

The phagocytic ability of carp (Cyprinus carpio L.) granulocytes was evaluated in vivo and in vitro. In suspensions of head kidney cells, neutrophil granulocytes incorporated both latex beads and coccidian merozoites. In intestinal tissues from carp with a Goussia carpelli infection, all granulocyte cell types (neutrophils and cells of the basophilic-eosinophilic complex) phagocytosed cell detritus and coccidian developmental stages, mainly merozoites.     

蒲公英糖蛋白体内外抗氧化作用研究

为了探讨蒲公英糖蛋白(TMGP)体内外抗氧化活性,采用H2O2、Fe2+、DPPH.和总抗氧化能力反应体系,检测蒲公英糖蛋白体外抗氧化活性,并与VC进行比较;同时建立D-半乳糖所致衰老小鼠模型,测定并比较TMGP组与模型组的血清、肝脏和脑内MDA含量和SOD、CAT、GSH-Px活性。结果显示:(1)随着TMGP浓度增加,其Fe2+、H2O2和DPPH.清除率均有所增加,当TMGP浓度为1.72mg/mL时,对H2O2、Fe2+清除率分别达到76.8%和55%,接近VC水平;当TMGP浓度为1.5mg/mL时,对DPPH.的清除率达到77%,也与VC水平相当,说明TMGP具有一定的体外总体抗氧化能力。(2)将TMGP按低、中、高剂量分别给小白鼠灌胃,可显著增强小鼠体内血清、肝脏、脑组织中SOD、CAT、GSH-Px活性,降低MDA含量,从而提高衰老小白鼠体内的抗氧化能力。研究表明,TMGP具有明显的体内外抗氧化作用。     

Tissue selective action of tamoxifen methiodide, raloxifene and tamoxifen on creatine kinase B activity in vitro and in vivo

We have compared the cell and tissue selective estrogenic and antiestrogenic activities of tamoxifen, raloxifene, ICI 164,384 and a permanently ionized derivative of tamoxifen — tamoxifen methiodide (TMI). This non-steroidal antiestrogen has limited ability to cross the blood brain barrier and is therefore less likely to cause the central nervous system disturbances caused by tamoxifen. We have used the stimulation of the specific activity of the “estrogen induced protein”, creatine kinase BB, as a response marker in bone, cartilage, uterine and adipose cells and in rat skeletal tissues, uterus and mesometrial adipose tissue. In vitro, TMI, tamoxifen and raloxifene mimicked the agonistic action of 17β-estradiol in ROS 17/2.8 rat osteogenic osteosarcoma, female calvaria, and SaOS₂ human osteoblast cells. In Ishikawa endometrial cancer cells, tamoxifen showed reduced agonistic effects and raloxifene showed no stimulation. However, as antagonists, tamoxifen and raloxifene were equally effective in Ishikawa or SaOS₂ cells. In immature rats, all four of the antiestrogens inhibited estrogen action in diaphysis, epiphysis, uterus and mesometrial adipose tissue; when administered alone, tamoxifen stimulated creatine kinase (CK) specific activity in all these tissues. Raloxifene and TMI, however, stimulated only the skeletal tissues and had no stimulatory effect in the uterus or mesometrial fat, and the pure antiestrogen ICI 164,384 showed no stimulatory effect in any of the tissues. The simultaneous injection of estrogen, plus an antiestrogen which acted as an agonist, resulted in lower CK activity than after injection of either agent alone. These differential effects, in vivo and in vitro, may point the way to a wider therapeutic choice of an appropriate antiestrogen which, although antagonizing E₂ action in mammary cancer, can still protect against osteoporosis and cardiovascular disease and not stimulate the uterus with its attendant undesirable changes, or interfere with the beneficial action of E₂ in the brain.     

pNEgr-mIL-12真核表达载体的体外和体内生物学活性检测

肿瘤的基因-放射治疗是近年来发展起来的新技术.分别于体外和体内检测含辐射敏感启动子和mIL-12基因的真核表达载体（pNEgr-mIL-12）的生物学活性.体外经酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)法检测转染pNEgr-mIL-12重组质粒的COS-7和B16细胞可经辐射诱导mIL-12 p70表达,于1.5～2.0 Gy照射后表达增高最明显,COS-7细胞于照后4 h 达峰值,B16细胞的表达水平随照射后时间延长而逐渐增高;pNEgr-mIL-12重组质粒联合电离辐射治疗小鼠移植肿瘤,单次或多次注射pNEgr-mIL-12重组质粒,联合局部照射能够抑制小鼠移植肿瘤生长,与单纯照射组比较肿瘤生长速度减慢,瘤重降低,尤以多次给予质粒治疗组效果明显.为进一步探讨最佳治疗方案及临床肿瘤病人的基因放射治疗提供了初步依据.     

Expression of thiol proteases decreases in tomato ovaries after fruit set induced by pollination or gibberellic acid

The modulation of the expression of thiol proteases during both senescence and development was investigated Proteolytic activity and some thiol proteases were analyzed in unpollinated tomato (Lycopersicon esculentum Mill. cv. Rutgers) ovaries during presenescence and during early fruit development induced by treatment with gibberellic acid (GA) or by natural pollination. Proteolytic activity in extracts was tested on azocasein and by observing degradation of the ribulose-1,5-bisphosphate carboxylase large subunit in western blots. There was no correlation between total activity and protein content. Thiol proteases were analyzed by western blot with antibodies raised against papain and a recombinant tomato C14 thiol protease. A 58-kDa polypeptide was recognized by both antibodies and two more polypeptides of 47 and 36 kDa were detected with the second one. All these polypeptide levels increased in untreated unpollinated ovaries at the presenescent stage. Natural pollination or GA treatment of unpollinated ovaries resulted in decreases of these polypeptides at an early developmental stage. The same pattern was observed for the levels of C14 mRNA. Our results suggest that the expression of C14 thiol protease occurs in unpollinated ovaries at the presenescent stage and that it can be suppressed by factors that induce fruit set and development.     

Partial characterization of a major gut thiol proteinase from larvae of Callosobruchus maculatus F.

Much of the proteolytic activity in the digestive tract of Callosobruchus maculatus larvae can be attributed to a thiol proteinase(s) that hydrolyzes [3H]methemoglobin optimally at pH 5.0. Maximal hydrolysis of [3H]methemoglobin, [3H]alpha-casein, and N-benzoyl-DL-arginine napthylamide-(BANA) required the presence of thiol reducing agents. Larval gut proteinase activity was strongly inhibited by p-hydroxymercuribenzoic acid (pHMB), Nethylmaleimide (NEM), and iodoacetic acid (IAA) but was unaffected by the Bowman-Birk and Kunitz proteinase inhibitors from soybeans or by lima bean trypsin inhibitor. L-Trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64), a specific inhibitor of thiol proteinases, potently inhibited proteolysis of [3H]methemoglobin by larval gut homogenates. Proteolytic activity in the larval gut was located in the lumen contents and thus appears to play a major role in extracellular digestion. The pH of the larval midgut is slightly acidic, and midgut contents exhibit a negative redox potential, conditions supporting the activity of a thiol proteinase. The significance of these findings is discussed with reference to the vulnerability of this digestive proteinase as a target for existing or genetically engineered plant chemical defenses.     

单增李斯特菌胎盘感染的体内外模型研究进展

单核细胞增生李斯特氏菌（Listeria monocytogenes）是重要的食源性致病菌,能引发人类的李斯特菌病,是全球公共卫生问题之一。该菌易感染孕妇,引起胎儿和新生儿的侵袭性李斯特菌病,严重威胁母婴健康。因此,建立有效的单增李斯特菌感染胎盘体内外模型,解析和探究单增李斯特菌经胎盘感染机制,是预防和控制单增李斯特菌感染母婴的关键所在。本文综述了可用于研究单增李斯特菌母婴感染的体内外胎盘模型,总结和讨论了各类模型的优势和局限性;并着重分析了体外三维胎盘屏障模型在单增李斯特菌感染方面的研究进展和未来研究方向。以期为深入解析该菌经胎盘感染的途径、发病机制提供支持,并为预防和控制母婴李斯特菌病提供科学参考。     

Comparative analysis of the sequences and structures of HIV-1 and HIV-2 proteases

The different isolates available for HIV-1 and HIV-2 were compared for the region of the protease (PR) sequence, and the variations in amino acids were analyzed with respect to the crystal structure of HIV-1 PR with inhibitor. Based on the extensive homology (39 identical out of 99 residues), models were built of the HIV-2 PR complexed with two different aspartic protease inhibitors, acetylpepstatin and a renin inhibitor, H-261. Comparison of the HIV-1 PR crystal structure and the HIV-2 PR model structure and the analysis of the changes found in different isolates showed that correlated substitutions occur in the hydrophobic interior of the molecule and at surface residues involved in ionic or hydrogen bond interactions. The substrate binding residues of HIV-1 and HIV-2 PRs show conservative substitutions of four residues. The difference in affinity of HIV-1 and HIV-2 PRs for the two inhibitors appears to be due in part to the change of Val 32 in HIV-1 PR to Ile in HIV-2 PR.     

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.