Suppression of the immune response to immunotoxins with anti-CD4 monoclonal antibodies.

Treatment of normal mice with a mAb to CD4 (GK1.5) was explored as a means of inhibiting the antibody response to an immunotoxin. Three days of pretreatment with 200 micrograms of GK1.5 completely abrogated the primary antibody response to a 3-micrograms dose of a mutant diphtheria toxin conjugated to an anti-transferrin receptor antibody. The same dose and schedule of anti-CD4 antibody significantly reduced and delayed, but did not prevent, the anamnestic antitoxin response in animals that had been previously primed to the immunotoxin. Three daily injections of anti-CD4 antibodies followed by weekly doses of immunotoxin resulted in a 3-wk delay in the development of antitoxin antibodies, and the kinetics of the antitoxin response correlated with the kinetics of recovery of CD4+ T cells in the spleen and lymph nodes. The antitoxin response to repeated doses of immunotoxin was completely abrogated when anti-CD4 antibodies were given every 2 wk throughout the course of immunotoxin treatment. Thus, transient depletion of Th cells during treatment can block the immune response to an immunotoxin. There was no evidence of tolerance induction with this regimen.     

转移及非转移肿瘤移植后615小鼠血液流变学变化的研究

血道高转移瘤株FC、淋巴合并血道高转移瘤株U14、淋巴道高转移瘤株H22、非转移瘤株P615分别接种于336只纯系近交615小鼠.不同时间取血并处死动物,进行组织学及血液流变学检查.将转移瘤发展过程分为潜伏期、侵袭期、转移早、中、晚期,非转移瘤发展过程分为潜优期、增殖期、囊腔形成期及中心坏死期.本实验结果显示,不同转移能力及途径肿瘤发展的不同时期血液流变学变化规律不同,因而表明肿瘤侵袭、转移与血液流变学变化之间存在互为因果的紧密关系.其临床诊断及治疗意义被讨论.     

基因定点诱变删除及天然α型心钠素的分泌型表达

用基因定点诱变技术,删除了pO_1α ANF表达质粒中的33对碱基,使人α型心钠素结构基因直接与大肠杆菌分泌型表达质粒pIN-Ⅲ-OmPA中的信号肽酶切位点编码区相连,构成天然人α型心钠素的表达质粒pANF,在IPTG诱导下表达28肽的天然人α型心钠素。纯化后的表达产物具有天然心钠素的放免活性和很强的舒张血管的生物活性。     

Incomplete fatty acid oxidation. The production and epimerization of 3-hydroxy fatty acids.

3-Hydroxydicarboxylic acids are major urinary metabolites derived from fatty acid metabolism. These compounds are produced from the omega-oxidation of 3-hydroxy fatty acids. The production of the precursor 3-hydroxy fatty acids from incomplete beta-oxidation of fatty acids in rat liver mitochondria was investigated. Independent of the chain length or the concentration of fatty acid substrates, the accumulation of 3-hydroxyacyl intermediates was relatively constant at the concentration of 3-5 nmol/mg of mitochondrial protein. The extent of the incomplete oxidation was the same in Percoll gradient-purified mitochondria. Rotenone treatment increased the production of 3-hydroxy fatty acids. 3-Hydroxy fatty acids did not exist as pure L-enantiomer as expected from beta-oxidation. Instead, these metabolites were epimerized to a near racemic mixture of D- and L-isomers with a slightly dominant D-isomer (58 +/- 3%). By using deuterium-isotope labeling, the mechanism of epimerizartion was shown to be a rapid dehydration-rehydration through trans-2-enoyl-CoA. In addition, cis-3 and trans-3 fatty acids were produced; these metabolites were derived from the isomerization of trans-2-enoyl-CoA. Epimerase and isomerase were thought to be enzymes involved in the oxidation of unsaturated fatty acids. Current data have shown that the metabolism of these acids is actually through NADPH-dependent reduction pathways. The activities of epimerase and isomerase detected in rat liver mitochondria possibly function mainly in the metabolism of saturated fatty acids in a reverse role to the conventional concept.     

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor): regulation of its production and possible roles in bone metabolism.

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor), expression of its mRNA, and possible roles in bone metabolism were studied in murine primary and clonal osteoblast-like cells. Local bone-resorbing factors such as IL-1, TNF alpha, and LPS strongly induced expression of LIF/D-factor mRNA in both clonal MC3T3-E1 cells and primary osteoblast-like cells. Neither parathyroid hormone nor 1 alpha,25-dihydroxyvitamin D3 stimulated expression of LIF/D-factor mRNA. LIF/D-factor per se did not stimulate expression of its own mRNA. Appreciable amounts of LIF/D-factor were detected in synovial fluids from rheumatoid arthritis (RA) patients but not in those with osteoarthritis (OA). Simultaneous treatment with LIF/D-factor, IL-1, and IL-6 at the concentrations found in synovial fluids from RA patients greatly enhanced bone resorption, though these cytokines did not stimulate bone resorption when separately applied. This suggests that LIF/D-factor produced by osteoblasts is in concert with other bone-resorbing cytokines such as IL-1 and IL-6 involved in the bone resorption seen in the joints of RA patients. LIF/D-factor specifically bound to MC3T3-E1 cells with an apparent dissociation constant of 161 pM and 1,100 binding sites/cell. LIF/D-factor dose-dependently suppressed incorporation of [3H]thymidine into MC3T3-E1 cells. In addition, it potentiated the alkaline phosphatase activity induced by retinoic acid, though LIF/D-factor alone had no effect on enzyme activity. These results suggest that LIF/D-factor is involved in not only osteoclastic bone resorption but also osteoblast differentiation in conjugation with other osteotropic factors.     

Components and proteolytic processing sites of arylsulfatase B from human placenta.

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.     

Utilization of the tricarboxylic acid cycle, a reactor design criterion for the microaerobic production of 2,3-butanediol

A new parameter, the relative utilization of tricarboxylic acid (TCA) cycle beta, is introduced to quantitatively account for the involvement of fermentation pathways and TCA cycle in the utilization of oxygen under oxygen-limiting (microaerobic) conditions. With the facultative anaerobe Enterobacter aerogenes, which produces 2,3-butanediol, a method is proposed to calculate beta from measurement of metabolites and exhaust gas. In continuous culture beta was found to be small under oxygen limitation, indicating that the fermentation pathways were preferred over the TCA cycle and oxygen was almost entirely consumed through oxidation of reduced nicotinamide adenine dinucleotide (NADH(2)) released by fermentation under these conditions. The increase of beta at high oxygen supply revealed a saturation of oxygen utilization through fermentation pathways. It could be concluded that, for the optimal performance of a microaerobic culture, oxygen uptake rate must be kept at such a level that as much NADH(2) as possible from fermentation pathways is oxidized by oxygen, and at the same time the utilization of TCA cycle is kept at a minimum. As the dynamics of the microaerobic culture can be fast, a significant effect of reactor hydrodynamics, i.e., mixing, on the overall performance can be expected. This was confirmed experimentally, and the parameter beta proved to be a useful reactor design criterium for the microaerobic cultivation. (c) 1992 John Wiley & Sons, Inc.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.

Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase (NOS). Herein, we report the molecular cloning of a cDNA encoding the constitutive calcium-calmodulin (Ca2+/CaM)-regulated nitric oxide synthase (ECNOS). A full-length ECNOS clone was isolated by screening a bovine aortic endothelial cell cDNA library using a fragment of rat brain NOS (bNOS) cDNA. This cDNA has an open reading frame of 3615 nucleotides encoding a 1205-amino acid protein. Membranes prepared from COS cells transfected with the ECNOS cDNA demonstrated NADPH- and Ca2+/CaM- dependent conversion of L-, but not D-, arginine to NO and citrulline that was inhibited by NG-nitro-L-arginine methyl ester. Comparison of the deduced amino acid sequence of ECNOS to the bNOS and macrophage NOS (Mac-NOS) sequences revealed 57 and 50% identity, respectively. In addition, ECNOS contains a unique N-myristylation consensus sequence (not shared by bNOS or Mac-NOS) that may explain its membrane localization.