Effect of covalent attachment of immunoglobulin fragments on liposomal integrity

Liposome stability during and after covalent coupling of Fab' antibody fragments was investigated. Large unilamellar vesicles containing entrapped 5(6)-carboxyfluorescein (CF) as a marker for liposomal integrity were prepared by extrusion through polycarbonate membranes. N-[4-(p-Maleimidophenyl)-butyryl]phosphatidylethanolamine (MPB-PE) was employed as a liposomal anchor for the covalent coupling of Fab' fragments. We observed that coupling of Fab' fragments to liposomes containing 5 mol % MPB-PE caused a concentration-dependent increase in size and polydispersity of the liposomes. Dependent on the concentration of the MPB-PE anchor in the membrane and the concentration of Fab' added, coupling was associated with the release of up to 95% of the entrapped CF. Rupture of the liposomes was identified as the primary mechanism of CF release during Fab' coupling. Reduction of the MPB-PE concentration to 1 mol % resulted in liposomes that were stable during and after Fab' coupling. The increased stability of these liposomes was due to the lower MPB-PE concentration and not to the lower number of attached Fab' fragments. By proper adjustment of the experimental conditions for coupling, the number of Fab' fragments attached to the 1 mol % MPB-PE liposomes could be increased without affecting the stability of the resulting liposomes. These stable liposomes, made by an extrusion method that avoids the use of organic solvents, detergents, or sonication, are therefore suitable for entrapment of labile compounds and can be used for immunotargeting or immunoassays.     

Suppressor T lymphocytes from lepromatous leprosy skin lesions

The immune response in leprosy forms a spectrum with lepromatous leprosy patients exhibiting specific unresponsiveness to antigens of Mycobacterium leprae. This unresponsiveness is thought to be related to the prevalence of T8-positive lymphocyte in these lepromatous lesions. To analyze the immunoregulatory function of these T8 cells, we developed simple procedures to extract lymphocytes from skin biopsy specimens of patients with leprosy. These lymphocytes were sorted for T8 and T4 positive cells, and cell lines were established by expansion with interleukin 2 (IL 2) and irradiated feeder cells. All T8 positive lines tested were positive for IL 2 receptors and HLA-DR determinants. These lines were additionally assayed for lepromin-induced suppression of the normal peripheral blood lymphocyte Con A proliferative response. Thirteen of 32 lines from six lepromatous patients showed significant suppressor activity, whereas nine lines from six tuberculoid patients and one line from normal peripheral blood failed to show suppression (p less than 0.001). Taken together, the finding of M. leprae-triggered suppressor cells within lepromatous skin lesions may in part explain the M. leprae unresponsiveness of lepromatous leprosy patients.     

Interleukin 1 activates phospholipase A2 in rabbit chondrocytes: a possible signal for IL 1 action

We examined the effects of Interleukin 1 (IL 1) on rabbit articular chondrocytes with particular emphasis on arachidonic acid metabolism in these cells. Articular chondrocytes were isolated from the knee joints of normal New Zealand white rabbits and were cultured in vitro until confluent. Addition of 5 U/ml of purified IL 1 to chondrocytes led to an early increase in cell-associated phospholipase A2 (PLA2; measured by hydrolysis of [14C]arachidonic acid-labeled E. coli). Within 1 hr after IL 1 addition, cell-associated PLA2 activity was increased by more than threefold relative to basal PLA2 activity, and further increases in cellular enzyme activity were observed up to 48 hr of IL 1 treatment. IL 1 stimulation also led to a time- and dose-related release of extracellular PLA2 and PGE2, but IL 1-induced PLA2 and PGE2 secretion occurred after the initial burst of intracellular PLA2 activity. Similar PLA2 and PGE2 responses were also observed when purified human IL 1 or IL 1-containing conditioned medium from LPS-stimulated human monocytes were used, but recombinant IL 2 or IL 3 were inactive. IL 1-induced chondrocyte PLA2 did not release radiolabeled free fatty acid from phosphatidylethanolamine labeled at the C-1 position with [14C]stearic acid, confirming the identity of this enzyme as PLA2. These data, therefore, provide the first direct evidence that IL 1 activates cellular PLA2, and we propose that PLA2 activation may be an early signal that initiates the inflammatory actions of IL 1.     

Syngeneic monoclonal internal image anti-idiotopes as prophylactic vaccines

A syngeneic monoclonal anti-idiotope that behaves as an internal image of the mammalian reovirus type 3 cellular attachment protein (viral hemagglutinin) was used in the syngeneic host for the induction of a prophylactic anti-viral antibody response. These studies were performed without the aid of co-stimulation by viral antigens. The high stringency of this system enables us to define the maximum constraints on the use of anti-idiotopes as anti-viral vaccines. We have used the murine BALB/c monoclonal IgM anti-idiotope 87.92.6 to study the idiotope and antigen specificity, kinetics, dose dependence, adjuvant, carrier, and valency requirements of anti-idiotope-induced anti-viral antibody responses. These studies show that the production of high titer neutralizing antibody requires a lengthy (60 day) immunization protocol, which includes the use of adjuvant and multivalent anti-idiotope, and is dependent on anti-idiotope concentrations of greater than 50 micrograms. When administered in this manner anti-idiotope can stimulate serotype-specific antibody responses across species barriers at levels comparable with those obtained after inoculation with virus. The practical efficacy of these reagents and procedures is documented by the ability of maternal immunization with anti-idiotope to confer complete protection in neonates from a potentially lethal reovirus type 3 viral infection.     

Starch phosphorylase inhibitor from sweet potato

A protein, starch phosphorylase inhibitor, was purified from the root of sweet potato (Ipomoea batatas [L.] Lam. cv Tainon 65). It had a molecular weight of 250,000 and could be composed of five identical subunits. The isoelectric point of the inhibitor was 4.63. It was a noncompetitive inhibitor toward the sweet potato enzyme with a K_i value of 1.3 × 10⁻⁶ molar when glucose-1-P was the variable substrate. Because cross-reacting materials of rabbit antiphosphorylase inhibitor of sweet potato were found in three arbitrarily selected plant materials, viz. potato tuber, spinach leaf, and rice grain, the occurrence of this protein seemed universal in higher plants. By an immunofluorescence technique, the inhibitor was located in the amyloplast and cell wall where phosphorylase was also found. This implies that they may interact in vivo, and the inhibitor may play an unknown regulatory role against the plant enzyme.     

Antithrombin III Basel. Identification of a Pro-Leu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity

Antithrombin III Basel is a hereditary abnormal antithrombin with normal progressive inhibition activity (normal reactive site) and reduced heparin cofactor activity (impaired heparin binding site). Structures of antithrombin III Basel and normal antithrombin III isolated from the same patient were compared by peptide mapping using the dimethylaminoazobenzene isothiocyanate precolumn derivatization technique. Of the approximately 50 tryptic peptides of normal and abnormal antithrombin III, one peptide comprising residues 40-46 had a different retention time in reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide from antithrombin III Basel had a single substitution of Pro (normal) by Leu (abnormal) at position 41. This substitution is close to an Arg (residue 47) and a Trp (residue 49) which have previously been shown to be critical for heparin binding by antithrombin III. Although additional amino acid substitutions in antithrombin III Basel cannot be ruled out, this Pro-Leu replacement could cause a conformational change by increasing both the helical structure and the hydrophobicity around residue 41. These data suggest that: (i) the heparin binding site of antithrombin III encompasses the region containing residues 41, 47, and 49; and (ii) the impaired heparin cofactor activity of antithrombin III Basel is likely due to a conformational change of the heparin binding site induced by the Pro-Leu substitution at position 41.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-bornyl pyrophosphate

Enzymes from Salvia officinalis and Tanacetum vulgare leaf epidermis catalyze the conversion of the acyclic precursor geranyl pyrophosphate to the cyclic monoterpenes (+)- and (-)-bornyl pyrophosphate, respectively. The antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H] Linalyl pyrophosphate (3H:14C = 5.22) was tested as a substrate with the cyclases from both sources to determine the configuration of the cyclizing intermediate. This substrate yielded (-)-bornyl pyrophosphate with 3H:14C ratio greater than 31, indicating specific utilization of (+)-(3S)-linalyl pyrophosphate as predicted. With the (+)-bornyl pyrophosphate cyclase, the 3H:14C ratio of the product was about 4.16, indicating a preference for the (-)-(3R)-enantiomer, but the ability also to utilize (+)-(3S)-linalyl pyrophosphate. (3R)- and (3S)-[1Z-3H]Linalyl pyrophosphate were separately compared to the achiral precursors [1-3H] geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. All functional precursors afforded optically pure (-)-(1S,4S)-bornyl pyrophosphate with the T. vulgare-derived cyclase (as determined by chromatographic separation of diastereomeric ketals of the derived ketone camphor), and (+)-(3S)-linalyl pyrophosphate was the preferred substrate. With the (+)-bornyl pyrophosphate cyclase from S. officinalis, geranyl, neryl, and (-)-(3R)-linalyl pyrophosphates gave the expected (+)-(1R,4R)-stereoisomer as the sole product, and (-)-(3R)-linalyl pyrophosphate was the preferred substrate. However, (3S)-linalyl pyrophosphate yielded (-)-(1S,4S)-bornyl pyrophosphate, albeit at lower rates, indicating the ability of this enzyme to catalyze the anomalous enantiomeric cyclization.     

二氧化硫熏气对小麦叶片中1-氨基环丙烷-1-羧酸、1-(丙二酰氨基)环丙烷-1-羧酸含量的影响及与乙烯产生的关系

在SO_2熏气9h过程中,小麦叶片中乙烯先上升,约6h达高峰,后下降;ACC含量则随熏气时间的延长而上升。停止熏气,乙烯继续下降,ACC含量也明显降低。MACC含量从熏气3h后不断上升,脱离接触后仍继续增加。6-BA预处理对SO_2引起的乙烯和ACC上升有促进作用,但对MACC含量无明显影响。SO_2熏气提高了乙烯形成酶活性。6-BA预处理对SO_2伤害有保护作用。对逆境乙烯的产生与调节作用进行了讨论。     

体外分化的人体巨噬细胞抑制天然杀伤(NK)细胞的研究

本文继先前工作后,进一步应用正常健康人外周血单个核细胞(PBMNC)经塑料培皿粘附技术把单核细胞分离出来,经培养进一步纯化,随后动态观察培养0,2,4,6和8天的单核-巨噬细胞的形态变化和对新鲜分离同种异基因个体PBMNC中NK活性的影响。实验表明,体外分化6天和8天的巨噬细胞质/核比例和胞浆内空泡显著增加,细胞直径约为0天时的2倍。这些细胞和PBMNC之比为0.5:1时,引起了NK细胞活性的50%以上抑制(4小时~(51)Cr标记K 562肿瘤的同位素释放试验)。这种抑制效应不为过氧化氢酶(Catalase 4000单位/毫升)和前列腺素合成酶的抑制剂(Indom 1×10~(-5)M)所阻断。实验证明,同种异基因个体的NK细胞不能识别巨噬细胞表面抗原,从而排除了巨噬细胞和K562肿瘤抗原竞争的可能性。实验还表明,巨噬细胞对NK活性的抑制是不受HLA约束的。应用高频超声振荡破碎巨噬细胞膜方法和免疫调变技术进一步提示,人体巨噬细胞对NK活性的抑制与巨噬细胞体积无关,而与体外分化所赋有的固有特性和它们分泌的免疫调节分子有关。     

Respiratory resistance with histamine challenge by single-breath and forced oscillation methods

Relaxed expirations were obtained from five anesthetized dogs under control conditions and during various rates of intravenous infusion of histamine. All volume vs. time curves obtained from 20 ms to 2 s after the start of expiration were poorly described by a single exponential function but were fitted very well by a biexponential function. The resistance of the respiratory system as a function of frequency from 2 to 26 Hz was also determined by the forced oscillation method in the same dogs. Three two-compartment models of the respiratory system were identified from the exponentials fitted to the relaxed expiration data, and the one that had the most plausible parameter values under control conditions consisted of a homogeneous lung compartment connected to a viscoelastic compartment. Although a two-compartment model is arguably appropriate for describing relaxed expirations in normal dogs, physiological considerations suggest that there should be more than two interacting components with histamine infusion. We cannot identify all these components from our data, however. The equivalent complex impedance of the respiratory system was also calculated from the biexponential curves and showed significant variation in resistance over the frequency range from 0 to 2 Hz and negligible variation above 2 Hz. The calculated resistances at 2 Hz were consistently higher than those obtained by the forced oscillation method, which may be due to the nonlinear behavior of the respiratory system during relaxed expiration. We conclude that the single-breath and forced oscillation methods should be viewed as providing complimentary information about respiratory resistance.