The ice-binding site of Atlantic herring antifreeze protein corresponds to the carbohydrate-binding site of C-type lectins

The type II antifreeze proteins (AFPs) of smelt and Atlantic herring are homologous to the carbohydrate-recognition domains (CRDs) of Ca2+-dependent (C-type) animal lectins and, like these lectins, acquire a stable and active structure upon binding Ca2+ ions. In the C-type lectin CRD, the carbohydrate-binding site is located at a Ca2+-binding site. Site-directed mutagenesis was used to test the hypothesis that the ice-binding site of the type II AFP corresponds to the carbohydrate-binding site of the lectins. To disrupt this site in the herring AFP without perturbing the Ca2+-dependent protein fold, a double mutant was constructed that changed the Ca2+- and carbohydrate-binding motif from the galactose-type of wild-type AFP containing the sequence Gln-Pro-Asp to a mannose-type that has the sequence Glu-Pro-Asn and is also known to bind Ca2+. The mutant AFP exhibited proper Ca2+ binding, folding, and stability as demonstrated by ruthenium red staining, proteolysis protection assays, and CD spectroscopy. However, it showed no antifreeze activity (thermal hysteresis) and did not alter ice crystal morphology to form bipyramidal crystals as does the active wild-type AFP. These results demonstrate that the ice-binding site of the herring type II AFP corresponds to the carbohydrate-binding site of the C-type lectin CRDs and further suggest that this ice-binding function evolved from the carbohydrate-binding site of a preexisting C-type lectin.     

Kynurenic acid distribution into brain and peripheral tissues of mice

Kynurenic acid (KYN), an antagonist of excitatory amino acid receptors, is a putative antidote against neuroexcitatory amino acid toxicity. We studied various doses (0.05-3.17 mmol/kg, i.p.) and the effects of probenecid coadministration (0.70 mmol/kg, i.p.) on tissue distribution of KYN in male and female Swiss-Webster mice. After injection of [3H]KYN, samples of brain, heart, liver, kidney, skeletal muscle, and gut were collected at selected times and assayed for KYN by liquid scintillation counting. The substance was absorbed rapidly and distributed into all tissues. Its content (nmol/g, mean +/- SE) at 60 min was 0.26 +/- 0.05, 1.80 +/- 0.05, and 40.4 +/- 8.1 in brain (for 0.05, 0.53, and 3.17 mmol/kg), 1.43 +/- 0.11, 14.3 +/- 3.7, and 212 +/- 32 in heart, 1.16 +/- 0.21, 10.6 +/- 2.6, and 254 +/- 21 in liver, and 7.41 +/- 2.65, 180 +/- 63, and 1899 +/- 254 in kidney. Net accumulation of KYN in brain was much lower than in other tissues. Probenecid increased KYN concentration in brain 2.5-fold. Peak brain:blood concentration ratio occurred between 60 and 180 min, was inversely associated with dose, and was not affected by probenecid. Although brain content was similar, female mice had an earlier peak brain:blood ratio (120 min) than males (180 min) for the 0.05 mmol/kg dose. Our results suggest the presence of a restricted transfer process for KYN with delayed egress from brain.     

Human papillomavirus in laryngeal and hypopharyngeal carcinomas. Relationship to survival

OBJECTIVE: To determine the relationship between human papillomavirus (HPV) DNA detection in upper aerodigestive tract malignancies and patient outcome. DESIGN: Archival paraffin-embedded specimens from 78 previously untreated patients with squamous carcinomas of the larynx and hypopharynx were pathologically verified and analyzed by polymerase chain reaction for detection of HPV DNA. Charts were independently reviewed and coded until final analysis. SETTING: The University of Texas M. D. Anderson Cancer Center, Houston, a tertiary cancer referral center. RESULTS: DNA was successfully extracted from 65 archival patient samples (83%). The mean (+/- SEM) duration of follow-up for these patients was 42 +/- 21 months. Thirty specimens (46%) exhibited detectable HPV DNA. Detection of HPV was significantly related to decreased survival, independent of disease stage. Log rank testing revealed that HPV detection, pathologic vascular invasion, and nodal status were the most significant predictors of death of disease. CONCLUSIONS: Laryngeal and hypopharyngeal carcinomas with detectable HPV may represent a biologically distinct subset of tumors that carry a poorer prognosis than do cancers with no detectable HPV.