Computed tomography detection of extracapsular spread of squamous cell carcinoma of the head and neck in metastatic cervical lymph nodes.

Background and purpose In patients with squamous cell carcinoma of the head and neck (HNSCC), extracapsular spread (ECS) of metastases in cervical lymph nodes affects prognosis and therapy. We assessed the accuracy of intravenous contrast-enhanced computed tomography (CT) and the utility of imaging criteria for preoperative detection of ECS in metastatic cervical lymph nodes in patients with HNSCC. Materials and methods Preoperative intravenous contrast-enhanced neck CT images of 93 patients with histopathological HNSCC metastatic nodes were retrospectively assessed by two neuroradiologists for ECS status and ECS imaging criteria. Radiological assessments were compared with histopathological assessments of neck dissection specimens, and interobserver agreement of ECS status and ECS imaging criteria were measured. Results Sensitivity, specificity, positive predictive value, and accuracy for overall ECS assessment were 57%, 81%, 82% and 67% for observer 1, and 66%, 76%, 80% and 70% for observer 2, respectively. Correlating three or more ECS imaging criteria with histopathological ECS increased specificity and positive predictive value, but decreased sensitivity and accuracy. Interobserver agreement for overall ECS assessment demonstrated a kappa of 0.59. Central necrosis had the highest kappa of 0.74. Conclusion CT has moderate specificity for ECS assessment in HNSCC metastatic cervical nodes. Identifying three or more ECS imaging criteria raises specificity and positive predictive value, therefore preoperative identification of multiple criteria may be clinically useful. Interobserver agreement is moderate for overall ECS assessment, substantial for central necrosis. Other ECS CT criteria had moderate agreement at best and therefore should not be used individually as criteria for detecting ECS by CT.

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme.

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu(2+) for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu(2+), indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu(2+) binding by RNA; rather, these data establish that Cu(2+) enters the active site within a Cu(2+)•GTPγS or Cu(2+)•GTP chelation complex, and that Cu(2+)•nucleobase interactions further enforce Cu(2+) selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg(2+) with [Co(NH3)6](3+) significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg(2+) coordination is structural rather than catalytic. Replacing Mg(2+) with alkaline earths of increasing ionic radii (Ca(2+), Sr(2+) and Ba(2+)) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.

A small ribozyme with dual-site kinase activity.

Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-(32)P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5' target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5' mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation.