Genetically tailored magnetosomes used as MRI probe for molecular imaging of brain tumor.

We investigate here the potential of single step production of genetically engineered magnetosomes, bacterial biogenic iron-oxide nanoparticles embedded in a lipid vesicle, as a new tailorable magnetic resonance molecular imaging probe. We demonstrate in vitro the specific binding and the significant internalization into U87 cells of magnetosomes decorated with RGD peptide. After injection at the tail vein of glioblastoma-bearing mice, we evidence in the first 2 h the rapid accumulation of both unlabeled and functionalized magnetosomes inside the tumor by Enhanced Permeability and Retention effects. 24 h after the injection, a specific enhancement of the tumor contrast is observed on MR images only for RGD-labeled magnetosomes. Post mortem acquisition of histological data confirms MRI results with more magnetosomes found into the tumor treated with functionalized magnetosomes. This work establishes the first proof-of-concept of a successful bio-integrated production of molecular imaging probe for MRI.

Equilibrium and kinetic parameters for the binding of inhibitors to the QB pocket in bacterial chromatophores: dependence on the state of QA.

The equilibrium and kinetic parameters for the binding of various inhibitors to the Q(B) pocket of the bacterial reaction center were investigated in chromatophores from Rhodobacter capsulatus and Rhodobacter sphaeroides. By monitoring the near-IR absorption changes specific to Q(A)(-) and Q(B)(-), we measured the fraction of inhibited centers in the dark and the kinetics and extent of inhibitor displacement after one flash due to the formation of the Q(A)Q(B)(-) state. The inhibitor release rate was much faster for triazines and o-phenanthroline (t(1/2) in the 50 ms to 1 s range) than for stigmatellin (t(1/2) approximately 20 s). For inhibitors with a rapid release rate, the fast phase of P(+) decay observed in the absence of secondary donor reflects the competition between P(+)Q(A)(-) recombination and inhibitor release: it is thus faster than the P(+)Q(A)(-) recombination, and its relative extent is smaller than the fraction of initially inhibited centers. At appropriate inhibitor concentrations, one can have almost total binding in the dark and almost total inhibitor displacement after one flash. Under such conditions, a pair of closely spaced flashes resets the two-electron gate in a single state (Q(A)Q(B)(-)), irrespective of the initial state. The apparent dissociation constant of terbutryn was significantly increased (by a factor of 4-7) in the presence of Q(A)(-), in agreement with the conclusion of Wraight and co-workers [Stein, R. R., et al. (1984) J. Cell. Biochem. 24, 243-259]. We suggest that this effect is essentially due to a tighter binding of ubiquinone in the Q(A)(-) state.

Absorption changes induced by the binding of triazines to the QB pocket in reaction centers of Rhodobacter capsulatus.

Inhibitors which block electron transfer from the primary (Q(A)) to the secondary (Q(B)) quinone of the bacterial reaction center are competing with the pool ubiquinones for binding at the Q(B) pocket. Due to the much greater stability of the semiquinone state Q(B)(-) compared with fully oxidized or reduced quinone, a displacement of the inhibitors takes place after one flash from state Q(A)(-)I to state Q(A)Q(B)(-). This process can be monitored from near-IR absorption changes which reflect local absorption shifts specific to Q(A)(-) and Q(B)(-). An anomalous behavior was observed when using triazines in chromatophores of R. capsulatus: the IR absorption change reflecting the formation of Q(B)(-) after one flash was absent. A normal transient decay of this signal was, however, triggered by a second flash, followed by a rapid return to the baseline. We show that this phenomenon is due to an absorption change induced by inhibitor binding (thus present in the dark baseline), with a spectrum close to that of Q(B)(-), so that the Q(B)(-) changes are canceled out during the inhibitor displacement process. On the second flash, one monitors the destruction of the semiquinone, leading transiently to the Q(A)Q(B) state, followed by inhibitor rebinding. This allows a direct measurement of the binding kinetics. This behavior was observed both in chromatophores and in isolated reaction centers from R. capsulatus, but not in R. sphaeroides.

Interactions between the donor and acceptor sides in bacterial reaction centers.

The apparent equilibrium constant K'(2) for electron transfer between the primary (Q(A)) and secondary (Q(B)) quinone acceptors of the reaction center was measured in chromatophores of Rhodobacter capsulatus. In the presence of the oxidized primary donor P(+), we obtained a value of K'(2)(P(+)) approximately 100 at pH 7.2, based on the rates of recombination from P(+)Q(A-) and P(+)Q(B-). K'(2) was also measured in the presence of reduced P, from the damping of semiquinone oscillations during a series of single turnover flashes. A 5-fold smaller value, K'(2)(P) approximately 20, was found. Additional information on the interactions between the donor and acceptor sides was obtained by measuring the shift of the midpoint potential of P caused by the presence of Q(B-) or Q(A-)S (where S indicates the presence of the inhibitor stigmatellin). A stabilization of the oxidized state P(+) was observed in both instances, by 10 mV for Q(B-) and 30 mV for Q(A-)S. The larger stabilization of P(+)Q(A-)S with respect to P(+)Q(B-) does not account for the effect of P(+)/P on K'(2). Analysis of these results indicates that the interactions between P(+)/P and Q(A)/Q(A)(-) are markedly modified depending on the occupancy of the Q(B) pocket by ubiquinone or by stigmatellin. We propose that the large value of K'(2)(P(+)) results essentially from a conformational destabilization of the P(+)Q(A-) state, that is relieved when the proximal site of the Q(B) pocket is occupied by stigmatellin.

Genotype determination at the survival motor neuron locus in a normal population and SMA carriers using competitive PCR and primer extension.

Precise quantitation of SMN1 copy number is of great interest in many clinical applications such as direct detection of SMA carriers or detection of an SMA-affected patient with a hemizygous deletion of the SMN1 gene. We describe a method that combines two independent nonradioactive PCR assays: determination of the relative ratio of the SMN1 and SMN2 genes using a primer extension assay and of the total SMN copy number using competitive PCR. Consistency of the results of two independent approaches ensures the reliability of the deduced genotype and thus avoids false interpretation of borderline results that can occur in quantitative assays. In all, 135 subjects were tested, including 91 normal controls and 44 SMA-affected children or SMA carriers. Two main genotypes were observed in controls: 2T/2C (45%) and 2T/1C (32%). A wide variability at the SMN locus is observed with nine different genotypes and up to six SMN genes. SMA carriers showed three frequent genotypes, 1T/2C (50%), 1T/3C (29%), and 1T/1C (18%). Normal chromosomes with two SMN1 genes per chromosome are not infrequent and thus, about 3% of SMA carriers are not detected using SMN1 copy number quantitation. Finally, as this method does not detect point mutations (4% of SMN1 gene mutations), reliability ranges from 93% to 100% depending on data available from the propositus.

Electron and proton transfer on the acceptor side of the reaction center in chromatophores of Rhodobacter capsulatus: evidence for direct protonation of the semiquinone state of QB.

1. The absorption changes associated with the formation of P+QBred (QBred stands for the semiquinone state of the secondary quinone acceptor) were investigated in chromatophores of Rhodobacter capsulatus. Marked modifications of the semiquinone spectrum were observed when the pH was lowered from 7 to 5. These modifications match those expected for a complete conversion of QBred from the anionic state QB- at pH 7 to the neutral protonated state QBH at pH 5. Similar modifications were observed in chromatophores from Rb. sphaeroides, but not in purified reaction centers from Rb. capsulatus, suggesting that the environment of the reaction center (native membrane vs detergent micelle) is the crucial parameter. 2. The recombination reaction P+QBred --> PQB was investigated as a function of pH. No particular kinetic heterogeneity was observed at low pH, showing that QBH remains mostly bound to the reaction center. The rate constant reaches a minimum value of 0.08 s-1 at pH 6, suggesting that the direct route for recombination prevails in chromatophores below this pH, instead of the usual pathway via QA-. 3. The proton uptake caused by QBred is about 1 below pH 7 and decreases at higher pH. It is suggested that the pH dependence of the conversion of QB- to QBH, occurring in a range where the uptake is constant, cannot be accommodated by a purely electrostatic model, but probably involves a conformational change. 4. The kinetics of the electron-transfer reaction QA-QB-->QAQBred were investigated. A 2-fold acceleration was observed between pH 7 and pH 5 (t1/2 approximately 30 and 15 microseconds, respectively). A fast (<<10 microseconds) unresolved phase appears to be present at both pHs. The second electron-transfer QA-QBred-->QAQBH2 proceeds with a similar rate as the first electron transfer (15-30 microseconds phase). Consequences for the rate-limiting step are discussed. 5. The carotenoid shift, indicative of the membrane potential, displays a rising phase concomitant with the QA-QB-->QAQBred electron transfer. Its relative extent is markedly increased at pH 5, with part of the kinetics occurring during the unresolved fast phase. 6. The extent of the electrochromic shift of bacteriopheophytin around 750 nm associated with formation of QBred decreases toward acidic pH, reflecting the charge compensation due to proton uptake and the formation of neutral QBH.

RET proto-oncogene mutations in French MEN 2A and FMTC families.

Constitutional mutations of the RET proto-oncogene have been identified in multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC) families. We sequenced RET exons 10 and 11 in 86 unrelated patients with an inherited predisposition to MTC (excluding MEN 2B). Germ-line mutations were identified in 93% of the MEN 2A families and 67% of the FMTC families tested. All were missense mutations affecting one of three cysteines in the extracellular domain of the RET tyrosine kinase receptor. The prevalence of phaeochromocytoma and hyperparathyroidism was significantly higher in families with a mutation of cysteine 634. These data confirm the preferential localisation of MEN 2A and FMTC associated mutations and the strong correlation between clinical manifestations and the position of RET mutation. Although direct sequencing of RET exons 10 and 11 allows the identification of a constitutional mutation in a large proportion of MEN 2A and FMTC families, our data sustain the existence of other MTC predisposing mutations elsewhere in RET coding or regulating region.