An actin-depolymerizing protein (destrin) from porcine kidney. Its action on F-actin containing or lacking tropomyosin

An Mr 19 000 protein (destrin) that has the ability to rapidly depolymerize F-actin in a stoichiometric manner was purified from porcine kidney by sequential chromatography on DNase I-agarose, hydroxyapatite, and Sephadex G-75. Its actin-depolymerizing activity is reversibly controlled by changes in KCl concentration but is insensitive to Ca2+ concentration. The rate of depolymerization of F-actin by destrin is much faster than that of spontaneous depolymerization induced by dilution and is not markedly decreased by the addition of end-blocking reagents such as cytochalasin B. These results suggest that destrin depolymerizes F-actin by interacting directly with F-actin protomers. Binding of muscle tropomyosin to F-actin slows down the rate of destrin-induced depolymerization of F-actin by about 30-fold. The data suggest that the destrin-induced depolymerization occurs from the ends of F-actin when F-actin is complexed with tropomyosin, but it takes place from the entire length of F-actin in the absence of tropomyosin.     

A possible clinical application of multicytokine-producing cytotoxic mononuclear cell (MCCM) therapy

When peripheral blood mononuclear cells (PBMC) were incubated with a streptococcal preparation, OK-432, for 24 h, PBMC acquired cytolytic activity against cultured and fresh human tumor cells. Such PBMC were called OK-432-activated mononuclear cells (OK-MC). OK-MC produce several kinds of cytokines such as interferon (IFN), IFN, and tumor growth inhibitory factor (TGIF) bothin vitro andin vivo. OK-MC-produced cytokines also inhibited the growth of cultured and fresh human tumor cells. The growth inhibition was examined by human tumor clonogenic assay using a double-layer agar technique. The results indicate that two pathways of anti-tumor activity are induced in OK-MC, i.e., cell-mediated and cytokine-mediated.     

Studies on the Flavanols in Tea

A method for the quantitative determination of flavanols in tea leaf and green tea is described. Separated flavanols on two-dimensional chromatogram are revealed by spraying dilute diazosulfanilic acid solution and eluted by hot water. To these eluates is added diazosulfanilic acid buffered with sodium acetate and after sufficient color development the absorbances of them are measured spectrophotometrically.     

ATP synthase--a marvellous rotary engine of the cell

ATP synthase can be thought of as a complex of two motors--the ATP-driven F1 motor and the proton-driven Fo motor--that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.     

Cl(-) concentration dependence of photovoltage generation by halorhodopsin from Halobacterium salinarum

The photovoltage generation by halorhodopsin from Halobacterium salinarum (shR) was examined by adsorbing shR-containing membranes onto a thin polymer film. The photovoltage consisted of two major components: one with a sub-millisecond range time constant and the other with a millisecond range time constant with different amplitudes, as previously reported. These components exhibited different Cl(-) concentration dependencies (0.1-9 M). We found that the time constant for the fast component was relatively independent of the Cl(-) concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl(-) concentrations. The fast and the slow processes were attributed to charge (Cl(-)) movements within the protein and related to Cl(-) ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that they corresponded to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl(-) concentration dependence, and the Cl(-) concentration dependence of its time constant suggested a weak and highly cooperative Cl(-)-binding site(s) on the cytoplasmic side (apparent K(D) of approximately 5 M and Hill coefficient > or =5). The Cl(-) concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be >100 ms.     

Induction of CD44 Variant 9-Expressing Cancer Stem Cells Might Attenuate the Efficacy of Chemoradioselection and Worsens the Prognosis of Patients with Advanced Head and Neck Cancer

Background

At our institute, a chemoradioselection strategy has been used to select patients for organ preservation on the basis of response to an initial 30–40 Gy concurrent chemoradiotherapy (CCRT). Patients with a favorable response (i.e., chemoradioselected; CRS) have demonstrated better outcomes than those with an unfavorable response (i.e., nonchemoradioselected; N-CRS). Successful targeting of molecules that attenuate the efficacy of chmoradioselection may improve results. Thus, the aim of this study was to evaluate the association of a novel cancer stem cell (CSC) marker, CD44 variant 9 (CD44v9), with cellular refractoriness to chemoradioselection in advanced head and neck squamous cell carcinoma (HNSCC).

Materials and Methods

Through a medical chart search, 102 patients with advanced HNSCC treated with chemoradioselection from 1997 to 2008 were enrolled. According to our algorithm, 30 patients were CRC following induction CCRT and 72 patients were N-CRS. Using the conventional immunohistochemical technique, biopsy specimens and surgically removed tumor specimens were immunostained with the anti-CD44v9 specific antibodies.

Results

The intrinsic expression levels of CD44v9 in the biopsy specimens did not correlate with the chemoradioselection and patient survival. However, in N-CRS patients, the CD44v9-positive group demonstrated significantly (P = 0.008) worse prognosis, than the CD44v9-negative group. Multivariate analyses demonstrated that among four candidate factors (T, N, response to CCRT, and CD44v9), CD44v9 positivity (HR: 3.145, 95% CI: 1.235–8.008, P = 0.0163) was significantly correlated with the poor prognosis, along with advanced N stage (HR: 3.525, 95% CI: 1.054–9.060, P = 0.0228). Furthermore, the survival rate of the CD44v9-induced group was significantly (P = 0.04) worse than the CD44v9-non-induced group.

Conclusions

CCRT-induced CD44v9-expressing CSCs appear to be a major hurdle to chemoradioselection. CD44v9-targeting seems to be a promising strategy to enhance the efficacy of chemoradioselection and consequent organ preservation and survival.     

Adenosine A(2A) receptors measured with [C]TMSX PET in the striata of Parkinson's disease patients

Adenosine A(2A) receptors (A2ARs) are thought to interact negatively with the dopamine D(2) receptor (D2R), so selective A2AR antagonists have attracted attention as novel treatments for Parkinson's disease (PD). However, no information about the receptor in living patients with PD is available. The purpose of this study was to investigate the relationship between A2ARs and the dopaminergic system in the striata of drug-na?ve PD patients and PD patients with dyskinesia, and alteration of these receptors after antiparkinsonian therapy. We measured binding ability of striatal A2ARs using positron emission tomography (PET) with [7-methyl-(11)C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([(11)C]TMSX) in nine drug-na?ve patients with PD, seven PD patients with mild dyskinesia and six elderly control subjects using PET. The patients and eight normal control subjects were also examined for binding ability of dopamine transporters and D2Rs. Seven of the drug-na?ve patients underwent a second series of PET scans following therapy. We found that the distribution volume ratio of A2ARs in the putamen were larger in the dyskinesic patients than in the control subjects (p<0.05, Tukey-Kramer post hoc test). In the drug-na?ve patients, the binding ability of the A2ARs in the putamen, but not in the head of caudate nucleus, was significantly lower on the more affected side than on the less affected side (p<0.05, paired t-test). In addition, the A2ARs were significantly increased after antiparkinsonian therapy in the bilateral putamen of the drug-na?ve patients (p<0.05, paired t-test) but not in the bilateral head of caudate nucleus. Our study demonstrated that the A2ARs in the putamen were increased in the PD patients with dyskinesia, and also suggest that the A2ARs in the putamen compensate for the asymmetrical decrease of dopamine in drug-na?ve PD patients and that antiparkinsonian therapy increases the A2ARs in the putamen. The A2ARs may play an important role in regulation of parkinsonism in PD.     

Adenosine A2A Receptors Measured with [11C]TMSX PET in the Striata of Parkinson's Disease Patients

Adenosine A_2A receptors (A2ARs) are thought to interact negatively with the dopamine D₂ receptor (D2R), so selective A2AR antagonists have attracted attention as novel treatments for Parkinson''s disease (PD). However, no information about the receptor in living patients with PD is available. The purpose of this study was to investigate the relationship between A2ARs and the dopaminergic system in the striata of drug-naïve PD patients and PD patients with dyskinesia, and alteration of these receptors after antiparkinsonian therapy. We measured binding ability of striatal A2ARs using positron emission tomography (PET) with [7-methyl-¹¹C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([¹¹C]TMSX) in nine drug-naïve patients with PD, seven PD patients with mild dyskinesia and six elderly control subjects using PET. The patients and eight normal control subjects were also examined for binding ability of dopamine transporters and D2Rs. Seven of the drug-naïve patients underwent a second series of PET scans following therapy. We found that the distribution volume ratio of A2ARs in the putamen were larger in the dyskinesic patients than in the control subjects (p<0.05, Tukey-Kramer post hoc test). In the drug-naïve patients, the binding ability of the A2ARs in the putamen, but not in the head of caudate nucleus, was significantly lower on the more affected side than on the less affected side (p<0.05, paired t-test). In addition, the A2ARs were significantly increased after antiparkinsonian therapy in the bilateral putamen of the drug-naïve patients (p<0.05, paired t-test) but not in the bilateral head of caudate nucleus. Our study demonstrated that the A2ARs in the putamen were increased in the PD patients with dyskinesia, and also suggest that the A2ARs in the putamen compensate for the asymmetrical decrease of dopamine in drug-naïve PD patients and that antiparkinsonian therapy increases the A2ARs in the putamen. The A2ARs may play an important role in regulation of parkinsonism in PD.     

One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar

F₁-ATPase is a rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α₃β₃-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the β-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F₁ rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F₁ molecules that carried a bead duplex on the γ-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 × 10⁷ M⁻¹s⁻¹. A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F₁ produced the same torque of ~40 pN·nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.