Frameless stereotaxy in a transmandibular, circumglossal, retropharyngeal cervical decompression in a Klippel-Feil patient: technical note.

Frameless stereotaxy, while most commonly applied to intracranial surgery, has seen an increasing number of applications in spinal surgery. Its use in the spine has been described to a greater degree in posterior rather than anterior surgical approaches, presumably due to the relative paucity of anatomical landmarks appropriate for frameless stereotactic registration in the anterior spine. This technical note illustrates the previously undescribed, successful use of frameless stereotaxy to the transmandibular, circumglossal, retropharyngeal surgical approach in a patient with Klippel-Feil syndrome.

Kinetic analysis of the nicking and hairpin formation steps in V(D)J recombination.

The complete cleavage phase of V(D)J recombination includes four phases: binding of the active RAG complexes to the 12- or 23-signals, nicking of the signals, synapsis of the two signals, and hairpin formation at both signals concurrently. We have done time courses for the complete cleavage phase of the V(D)J recombination reaction and quantitated the amount of active RAG enzyme. We have also formulated a kinetic model for the binding, nicking, synapsis, and hairpin formation phases. We have utilized free solution enzymatic measurements for the binding and nicking phases as we do mathematical simulations of the kinetic model. This permits iteration of rate constants for the synapsis and hairpin formation phases until the model fits the observed overall cleavage time course. This process yields a rate constant for the hairpin formation that is 0.004 min(-1), which corresponds to an average catalytic cycle time of 250 min. This value is exceedingly close to a measured value of this constant that relied on wash-out of an inhibitory cofactor. The agreement indicates that this is likely to be the rate of the hairpin step over a wide range of range of conditions and irrespective of the DNA sequence of the V, D or J coding end located adjacent to the signal. These findings indicate that, under optimal in vitro conditions, the core RAG proteins carry out nicking at a rate which is nearly 150-fold faster than hairpin formation. The physiologic implications of this and other kinetic inferences of these time courses are discussed.

The cleavage efficiency of the human immunoglobulin heavy chain VH elements by the RAG complex: implications for the immune repertoire.

The human immunoglobulin heavy chain locus contains 39 functional human V(H) elements. All 39 V(H) elements (with their adjacent heptamer/nonamer signal) were tested for site-specific cleavage with purified human core RAG1 and RAG2, and HMG1 proteins in a 12/23-coupled cleavage reaction. Both nicking and hairpin formation were measured. The individual V(H) cleavage efficiencies vary over nearly a 30-fold range. These measurements will be useful in considering the factors affecting the generation of the immunoglobulin and T-cell receptor repertoires in the adult humans. Interestingly, when these cleavage efficiencies are summed for each of the V(H) families, the six V(H) family efficiencies correspond closely to the observed profile of unselected V(H) family usage in the peripheral B cells of normal adult humans. This correspondence raises the possibility that the dominant factor determining V(H) element utilization within the 1-megabase human genomic V(H) array is simply the individual RAG cleavage efficiencies.