Influence of zoledronic acid on disseminated tumor cells in primary breast cancer patients.

BACKGROUND: The presence of disseminated tumor cells (DTCs) in bone marrow of patients with early breast cancer (EBC) has been correlated with increased risk of metastatic disease or locoregional relapse. Zoledronic acid (ZOL) treatment has reduced DTCs in the bone marrow of patients with EBC in several studies. This controlled study sought to confirm these observations. PATIENTS AND METHODS: Patients with EBC and DTC-positive bone marrow were randomized (N = 96) to treatment with ZOL plus adjuvant systemic therapy or adjuvant systemic therapy alone. The change in DTC numbers at 12 months versus baseline was measured. RESULTS: DTC-positive patients treated with ZOL were more likely to become DTC-negative after 12 months of treatment compared with the controls (67% versus 35%; P = 0.009). At 12 months, DTC counts decreased to a mean of 0.5 ± 0.8 DTCs in the ZOL group and to 0.9 ± 0.8 DTCs in the control group. In addition, ZOL was generally well tolerated. CONCLUSIONS: Treatment with ZOL improves elimination of DTCs. Further studies are needed to determine whether the reduction in DTCs by ZOL provides clinical benefit.

Functional citric acid cycle in an arcA mutant of Escherichia coli during growth with nitrate under anoxic conditions.

The operation of the citric acid cycle of Escherichia coli during nitrate respiration (anoxic conditions) was studied by measuring end products and enzyme activities. Excretion of products other than CO2, such as acetate or ethanol, was taken as an indication for a non-functional cycle. From glycerol, approximately 0.3 mol acetate was produced; the residual portion was completely oxidized, indicating the presence of a partially active citric acid cycle. In an arcA mutant devoid of the transcriptional regulator ArcA, glycerol was completely oxidized with nitrate as an electron acceptor, demonstrating derepression and function of the complete pathway. Glucose, on the other hand, was excreted mostly as acetate by the wild-type and by the arcA mutant. During growth on glucose, but not on glycerol, activities of succinate dehydrogenase and of 2-oxoglutarate dehydrogenase were missing nearly completely. Thus, the previously described strong repression of the citric acid cycle during nitrate respiration occurs only during growth on glucose and is the effect of anaerobic and, more important, of glucose repression. In Pseudomonas fluorescens (but not Pseudomonas stutzeri), a similar decrease of citric acid cycle function during anaerobic growth with nitrate was found, indicating a broad distribution of this regulatory principle.

Growth phase-dependent regulation of nuoA-N expression in Escherichia coli K-12 by the Fis protein: upstream binding sites and bioenergetic significance.

The expression of the nuoA-N operon of Escherichia coli K-12, which encodes the proton-pumping NADH dehydrogenase I is modulated by growth phase-dependent regulation. Under respiratory growth conditions, expression was stimulated in early exponential, and to a lesser extent in late exponential and stationary growth phases. The stimulation in the early exponential growth phase was not observed in fis mutants, which are deficient for the growth phase-responsive regulator Fis. Neither the alternative sigma factor RpoS nor the integration host factor (IHF) are involved in growth phase-dependent regulation of this operon. When incubated with nuo promoter DNA, isolated Fis protein formed three retarded complexes in gel mobility experiments. DNase I footprinting identified three distinct binding sites for Fis, 237 bp (fis1), 197 bp (fis2) and 139 bp (fis3) upstream of the start of the major transcript of nuoA-N, T1. The protein concentrations required for half-maximal binding to fis1, fis2 and fis3 were about 20 nM, 40 nM and 100 nM Fis, respectively. The IHF protein bound 82 bp upstream of the start of transcript T2 with a half-maximal concentration for binding of 50 nM. Due to the growth phase-dependent regulation by Fis, the synthesis of the coupling NADH dehydrogenase I is increased relative to that of the noncoupling NADH dehydrogenase II during early exponential growth. This ensures higher ATP yields under conditions where large amounts of ATP are required.

Control of FNR function of Escherichia coli by O2 and reducing conditions.

The synthesis of the enzymes constituting the electron transport chain of Escherichia coli is controlled by electron acceptors in order to achieve high ATP yields and high metabolic rates as well. High ATP yields (or efficiency) are obtained by the use of electron acceptors for respiration which allow high ATP yields, preferentially O2, and nitrate in the absence of O2. The rate of metabolism is adjusted by use of respiratory isoenzymes which differ in the rate and the efficiency of energy conservation, such as the non-coupling NADH dehydrogenase II (ndh gene) and the coupling NADH dehydrogenase I (nuo genes). By combination of the contrary principles (rate versus efficiency), growth is optimized for growth yields and rates. One of the major transcriptional regulators controlling the switch from aerobic to anaerobic respiration is FNR (fumarate nitrate reductase regulator). FNR is located in the cytoplasm and contains a [4Fe-4S] cluster in the active (anaerobic) state. By reaction with O2 the cluster is converted to a [2Fe-2S] cluster and finally to apoFNR. O2 diffuses into the cytoplasm even at very low O2-tensions (1 microM) where it inactivates [4Fe-4S] x FNR. The formation of [4Fe-4S] x FNR from apoFNR can use glutathione as a reducing agent in vitro. This process could also be important for the reductive activation of FNR in vivo. A model for the control of the functional state of FNR by O2 and glutathione is discussed. According to this model the functional state of FNR is determined by a (rapid) inactivation of FNR by O2, and a slow (constant) reactivation with glutathione as the reducing agent.

LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli.

The function of the LysR-type regulator LrhA of Escherichia coli was defined by comparing whole-genome mRNA profiles from wild-type E. coli and an isogenic lrhA mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80. When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant. In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent K(D) approximately 20 nM), whereas the promoters of fliC, fliA and trg were not bound by LrhA. The expression of flhDC (encoding FlhD(2)C(2)) was derepressed by a factor of 3.5 in the lrhA mutant. FlhD(2)C(2) is known as the master regulator for the expression of flagellar and chemotaxis genes. By DNase I footprinting, LrhA binding sites at the flhDC and lrhA promoters were identified. The lrhA gene was under positive autoregulation by LrhA as shown by gel retardation and lrhA expression studies. It is suggested that LrhA is a key regulator controlling the transcription of flagellar, motility and chemotaxis genes by regulating the synthesis and concentration of FlhD(2)C(2).