Feedback inhibition of homoserine kinase from radish leaves

Homoserine kinase is a potential control point in the biosynthetic pathway for threonine, isoleucine and methionine. The radish leaf enzyme was tested     

Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TG^r mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrd^r mutations showed a curvilinear dose-response curve. The BrdUrd^r frequency was roughly proportional to the square of the TG^r frequency and the results were compatible with the hypothesis that BrdUrd^r cells were induced by two mutational events within a cell. Most of the BrdUrd^r colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrd^r colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells.

Reverse selection from BrdUrd^r to HAT^r was accomplished with frequencies of 10⁻⁶−10⁻³ for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10⁻¹. Further re-revertants from HAT^r to BrdUrd^r were also found with frequencies of 10⁻³−10⁻² without treatment. These results indicate that the initial BrdUrd^r cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.     

Effect of auxin on the metabolism of mevalonic acid in suspension-cultured carrot cells

The rate of incorporation of [¹⁴C]mevalonate into carotenoid and steroid fractions in suspension-cultured carrot cells decreased markedly after 2,4-dichlorophenoxyacetic acid was removed from the medium. In parallel to this change, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a microsomal fraction was reduced to ca 33% of the control value, while that of a particulate fraction showed no significant change. The activities of mevalonate activating enzymes remained unchanged after auxin deprivation.     

Comparative effectiveness of lymphotoxin anticarcinogenic and tumor cell growth-inhibitory activities

Prevention of ultraviolet radiation- or chemical carcinogen-induced morphologic transformation and inhibition of tumor-producing transformed cell growth by lymphotoxin and by normal spleen leukocytes were quantitatively compared to define the antineoplastic activity spectra of these natural immune mediators. When Syrian golden hamster embryo cells seeded for colony formation in culture dishes were treated simultaneously with carcinogen and lymphotoxin, the number of morphologically transformed cell colonies was irreversibly reduced by 50% in the presence of 6 units of lymphotoxin/ml. Lymphotoxin inhibition of tumor cell growth, however, was reversible and 50% reduction in tumor cell growth in three transformed lines required 124, 330, and 477 units/ml. Thus, the anticarcinogenic activity of lymphotoxin can be 20-fold or more greater than its tumor growth-inhibitory activity. Similarly spleen leukocytes also were more effective as an anticarcinogen than as an inhibitor of tumor cell growth, consistent with previous observations that naturally occurring spleen leukocyte antineoplastic activity may result from lymphotoxin secretion.     

Lymphocyte transformation to connective tissue antigens in adult and juvenile rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus, and a nonarthritic control population

Transformation of peripheral blood lymphocytes after exposure to connective tissue antigens was measured in patients with adult (n = 35) and juvenile rheumatoid arthritis (n = 34), osteoarthritis (n = 21), ankylosing spondylitis (n = 15), and systemic lupus erythematosus (n = 26) and in control subjects (n = 36). The connective tissue antigens included homologous cartilage-type proteoglycan, cyanogen bromide-derived peptides of type I, II, and III collagens, and type I and II helical collagens. Lymphocyte transformation was not detected in the osteoarthritic and control groups, with one exception. Sensitization to at least one connective tissue antigen was detected in approximately one-third of the rheumatoid arthritic and lupus patients and in one-quarter of the juvenile rheumatoid patients. In ankylosing spondylitis, positive responses occurred to proteoglycan in 20% of patients tested but never to collagens or peptides. Sensitivity to proteoglycan was detected only in ankylosing spondylitis except for one patient with juvenile rheumatoid arthritis. In patients with systemic lupus erythematosus and both forms of rheumatoid arthritis, lymphocyte transformation was usually more frequently detected to peptides than to the helical collagens. In adult rheumatoid arthritis, type II peptides elicited an elevated number of responses (14%) as did type I (9%) and III (8%) peptides to lesser degrees. Responses to type I (4%) and II (4%) helical collagens were infrequent. Rheumatoid arthritic patients usually exhibited sensitivity to only one antigen and lymphocyte transformation was often detected when the arthritis was improving. In juvenile rheumatoid arthritis, lymphocyte transformation was detected to peptides of type I (16%), II (9%), and III (29%) collagens and to helical type I (12%) and II (8%) collagens. In systemic lupus erythematosus, sensitization was detected to peptides of type I (13%), II (20%), and III (14%) collagens and to helical type I collagen (18%) but not type II collagen. Simultaneous sensitivity to several antigens often occurred in both systemic lupus erythematosus and juvenile rheumatoid arthritis. Examination of individual patients in all three rheumatic disease groups revealed that immune sensitivity developed to collagen peptides rather than to the helical molecules, particularly in the case of type II collagen. Thus, some patients with inflammatory arthritis exhibit immune responses to connective tissue components which are, as a group, characteristic for each type of arthritis. These responses, which were not obviously associated with disease activity, may develop as a result of inflammation or trauma which destroys connective tissue and exposes molecules, in either a native or degraded state, to cells of the immune system. Expression of sensitivity to these tissue antigens may contribute to the chronicity of the inflammatory arthritides.     

Intercellular communication in rat seminiferous tubules

Intercellular electrical coupling in seminiferous tubules from prepubescent and adult Wistar rats has been studied by using conventional techniques. It is found that cells in the seminiferous epithelium are electrically coupled. Experiments performed using "Sertoli cell-enriched" seminiferous tubules indicate the existence of intercellular ionic communication between Sertoli cells. Junctional conductance is independent of the direction of electrical field and it is affected by A23187 Ca ionophore (5 microM) but not by exposure to the neurotransmitter norepinephrine (1-5 X 10(-5) M). Intracellular resistivity (including junctional resistance) is higher in mature as compared to immature germinal epithelium. These findings suggest that cell metabolites or second messenger molecules could be transferred via the low-resistance pathways between epithelium cells to coordinate cellular activity.     

人胃癌不同周期细胞膜表面ConA受体的分布与侧向运动

本文研究了人胃低分化粘液性腺癌细胞MGC 80-3不同周期时相中ConA受体的分布与侧向运动。MGc 80-3细胞经同步化培养,用F-ConA标记。被标记细胞中G_1、S和G_2期呈不连续的分布,但它们之间又存在显著的差异。M期呈较均匀的强荧光分布(与其它时相细胞比较)。荧光漂白恢复方法测定ConA受体复合物侧向运动表明:各个周期时相之间不仅运动方式不同,而且运动速率也有显著差异。M期与G_1期主要表现出扩散型运动;而S期与G_2期表现为流动型运动。G_1期的扩散系数大干M期的;S期的流动速率大于G_2期的。但可动分子百分比以G_2期最高。这些结果表明了ConA受体的动力学性质。它受到细胞周期的调节。     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and I h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED₅₀s of these antitumor effects afteri.v. administration of rHu-TNF were about 50 times as high as ED₅₀s afteri.t. administration. ED₅₀ ofi.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED₅₀ ofi.v. rHu-TNF for vascular effect was only 2–3 times as high as that for cytotoxicity. The whole body autoradiographies with [¹²⁵I] HSA giveni.v. to see the blood influx into tumor tissue and [¹⁴C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues.In conclusion, thein vivo antitumor effect of rHu-TNF giveni.t. ori.v., appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.