Energy metabolism and blood perfusion in a mouse mammary adenocarcinoma during growth and following X irradiation

Biochemical and blood perfusion changes in a mouse tumor system (MDAH MCaIV) were studied relative to normal tissues under conditions of normal blood flow and clamped blood supply. Further studies were performed during tumor growth and after local X irradiation. The biochemical profiles of three untreated human soft tissue sarcomas were also investigated. Animal tumors were irradiated in situ with either a single or fractionated regime to total doses of 20 or 49 Gy. Assays of lactate, pyruvate, AMP, ADP, and ATP were made on freeze-clamped tissue following authentic or sham treatments. Blood perfusion to tumors treated in the same way was measured using iv injection of 201Tl. The human tumors were found to have a lower lactate to pyruvate ratio (L/P) than the MCaIV tumors; their ATP levels were also lower. L/P was much higher in the MCaIV tumors than in normal liver, kidney, and muscle in the mouse. Occlusion of the blood supplies of the normal kidney and the MCaIV tumor caused an increase in the lactate and L/P levels in both cases. However, whereas the ATP level in the kidney fell, the level in the tumor was maintained. There was some evidence that the adenine nucleotides were not in equilibrium via the adenyl kinase catalyzed reaction. In addition, tumors were found to contain the enzyme creatine kinase. These results suggest that energy charge calculations cannot be computed in a meaningful manner because the creatine kinase catalyzed phosphorylation of ADP would maintain a higher than normal ATP level. Lactate and L/P ratio was found to increase during tumor growth and decrease following X irradiation. The total adenine nucleotides (AMP + ADP + ATP) exhibited a trend toward lower values with increasing tumor size. There was no significant change in total adenine nucleotides after a single 20-Gy dose; however, fractionated radiation caused some fall in total nucleotides. It is concluded that, in this tumor system, lactate level is a sensitive index of radiation-induced biochemical changes which are likely to reflect changes in tumor oxygenation.     

Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis

The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Column centrifugation generates an intersubunit disulfide bridge in Escherichia coli F1-ATPase

Passage of F1-ATPase through a centrifuge column [Penefsky, H. S. (1979) Methods Enzymol. 56, 527-530] caused formation of a product with a relative molecular mass of 72,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The product was identified as cross-linked alpha and delta subunits by using Western blots and subunit-specific monoclonal antibodies. The cross-link was reversed by 50 mM dithiothreitol implying that it was a disulfide bridge. Formation of the cross-link was inhibited by 2 mM EDTA and was stimulated in some buffers by the addition of 10 microM CuCl2. Time course experiments indicated that the majority of the cross-link formed while the enzyme was passing through the column. Thus the cross-link induced by column centrifugation arose from the rapid, heavy-metal-ion-catalysed oxidation of two sulfhydryl groups, one on the alpha subunit and one on the delta subunit, to a disulfide. These results demonstrate that care must be exercised when running proteins through centrifuge columns as potentially deleterious disulfide formation can result. An anti-beta monoclonal antibody was capable of immunoprecipitating the entire enzyme including the cross-linked subunits, implying that the cross-linked alpha and delta subunits were still a part of F1. The formation of the cross-link affected neither the hydrolytic activity of the enzyme nor its susceptibility to inhibition by epsilon subunit. The cross-linked enzyme was unable to bind to F1-depleted membranes in experiments in which soluble F1 and membranes were separated by centrifugation. Column centrifugation did not generate the cross-link on membrane-bound enzyme. These results indicate that the alpha-delta cross-link results in a loss of binding affinity between F1 and F0.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

Mechanisms of cytotoxicity induced by horseradish peroxidase/indole-3-acetic acid gene therapy

We have previously proposed the horseradish peroxidase (HRP) and the non-toxic plant hormone indole-3-acetic acid (IAA) as a novel system for gene-directed enzyme/prodrug therapy (GDEPT). The cytotoxic potential of HRP/IAA GDEPT and the induction of a bystander effect were demonstrated in vitro under normoxic as well as hypoxic tumour conditions. To date, the chemical agents and the cellular targets involved in HRP/IAA-mediated toxicity have not been identified. In the present work, some of the molecular and morphological features of the cells treated with HRP/IAA gene therapy were analysed. Human T24 bladder carcinoma cells transiently transfected with the HRP cDNA and exposed to the prodrug IAA showed chromatin condensation, formation of apoptotic bodies, DNA fragmentation, and Annexin V binding. Similar effects were observed when the cells were incubated with the apoptotic agent cisplatin. Caspases appeared to be involved as effectors in HRP/IAA-mediated apoptosis, since treatment with a general caspase inhibitor decreased the fraction of cells with micronuclei (MN) by 30%, with fragmented DNA by 50%, and with condensed chromatin by 60%. However, very little degradation of one of the downstream targets of caspase-3, PARP, could be detected, and apoptosis alone did not appear to account for the killing levels measured with a clonogenic assay. The effect of HRP/IAA treatment on cell cycle progression was also investigated, and a rapid cytostatic effect, equally affecting all phases of the division cycle, was observed.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.