Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

Cryptosporidium sp. Infections in Green Turtles, Chelonia mydas, as a Potential Source of Marine Waterborne Oocysts in the Hawaiian Islands.

For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum.

Resolving receptor heterogeneity using Fourier-derived affinity spectrum analysis and LIGAND: benzodiazepine receptors in the rat spinal cord.

In the present study, we combined a powerful and novel receptor binding data analysis technique. Fourier-derived affinity spectrum analysis (FASA), with the nonlinear regression analysis program LIGAND to resolve benzodiazepine receptor heterogeneity in rat spinal cord. With FASA, we identified three distinct [3H]Ro15-1788 binding populations: two high-affinity sites (0.4 and 5 nM) for the radioligand and a lower-affinity site (150 nM) that is insensitive to the imidazopyridine alpidem. With the affinities for the radioligand determined with FASA, the Ki values of 13 competing ligands were calculated with LIGAND. All of the ligands studied displayed the highest affinity for site 1 (the highest-affinity [3H]Ro15-1788 binding site), with the exception of AHR 11797. Site 2 had high affinity for Ro15-1788, lower affinity for flunitrazepam and beta-CCM and very low, but measurable, affinity for zolpidem. The alpidem-insensitive binding site, studied in isolation by performing competitive binding assays in the presence of 65 microM alpidem, showed relatively low affinity for all of the ligands studied, and its physiological relevance is not yet known.

Chronic anxiolytic treatment effects on conflict behavior in the rat.

The present studies examined the effects of chronic posttest treatment with the antipanic agent alprazolam (ALP) or the traditional anxiolytic agents chlordiazepoxide (CDP) and phenobarbital (PhB) on conflict behavior. In daily ten-minute sessions, water-deprived rats were trained to drink from a tube which was occasionally electrified (0.25 or 0.5 mA). Electrification was signalled by a tone. Chronic ALP (10 mg/kg/day), CDP (40 mg/kg/day), PhB (80 mg/kg/day) or vehicle were injected IP after conflict testing (in some experiments again 12-16 hours later) for a minimum of 6 weeks. Chronic ALP (but not CDP or PhB) resulted in a time-dependent increase in punished responding, with a latency to onset of 3-4 weeks; this effect was not antagonized by the benzodiazepine antagonist Ro15-1788. These data support the hypothesis that conflict paradigms may serve as animal models for the study of antipanic agents. Moreover, these data suggest that not all anxiolytics will exhibit antipanic efficacy.

Chronic antidepressant and clonidine treatment effects on conflict behavior in the rat.

The present studies examined the effects of chronic treatment with several antidepressants and clonidine on conflict behavior. In daily ten-minute sessions, water-deprived rats were trained to drink from a tube which was occasionally electrified (0.25 or 0.5 mA). Electrification was signalled by a tone. Chronic desipramine (5 mg/kg, IP, b.i.d.) or clonidine (40 micrograms/kg, b.i.d.) treatment resulted in time-dependent anticonflict effects, with a latency to onset of approximately 3-4 weeks. In contrast, chronic buproprion (up to 10 mg/kg, IP, b.i.d.), mianserin (up to 10 mg/kg, IP, b.i.d.) or trazodone (up to 40 mg/kg, IP, b.i.d.) treatment resulted in at best only a weak anticonflict effect. The efficacy of these antidepressants and clonidine to increase punished responding when administered chronically correlates well with their efficacy as antipanic agents in man.

Predicting discharge and follow-up status of hospitalized adolescents.

The functioning of 58 adolescent psychiatric patients at hospital admission, discharge, and one-and-a-half-year follow-up was examined in relation to six predictor variables: level of precipitating stress, primary process thinking on the Rorschach test, cognitive inefficiency, severity of psychopathology, maladaptive behavior on the unit, and process versus reactive illness. Severity of psychopathology and cognitive inefficiency were significantly related to the adolescents' functioning at all three observation points. High precipitating stress and short duration of symptoms (that is, in reactive illnesses) were associated with significantly better functioning at follow-up. Primary process thinking on the Rorschach was correlated with severe disturbances on admission, but was unrelated to outcome at discharge or follow-up. Maladaptive behavior on the unit was unrelated to functioning at any time. The authors concluded that outcome for hospitalized adolescents is determined by key patient differences and is systematically predictable.

Quantification and simulation of fluid-filled catheter/transducer systems.

Optimization of fluid-filled catheter/transducer systems for blood pressure measurement requires a prior knowledge of how each component affects overall dynamic response. This paper presents a method to measure resistance, inertance, and compliance of individual components by investigating the causal relationships between added known compliances and resonance. Additionally, a computer simulation that combines individual component values to synthesize practical blood pressure monitoring systems is presented. Complete monitoring systems are modeled through the combination of characterized components such as transducers, monitoring lines, and catheters and used to predict overall system response to any input function.

Location of the Bacillus subtilis temperate bacteriophage phi 105 attP attachment site.

Chromosomal DNAs of lysogens of phi 105 and phi 105 DI:1t were digested with restriction enzymes EcoRI and HpaI and were probed with nick-translated mature phi 105 DNA. Altered bacteriophage-specific bands in the lysogens were detected, indicating that the phage integrates into the host chromosome at a single site, probably via a Campbell-type circular intermediate. The phage attachment site is centrally located in the phage genome and lies between the phage immunity region and the nonessential deletable region of phi 105.

Nucleotide sequence of the cohesive single-stranded ends of Bacillus subtilis temperate bacteriophage phi 105.

The cohesive single-stranded ends of temperate Bacillus subtilis phage phi 105 were analyzed with the exonuclease activities of the Klenow fragment of DNA polymerase I and with exonuclease III and were found to be 3' extensions. Chemical sequencing of 3'-end-labeled fragments showed that the ends are 7-base extended 3' single strands and have the sequence: 5'-GCGCTCC-3'. 3'-CGCGAGG-5'

Interpretation of beat-to-beat blood pressure values in the presence of ventilatory changes.

When blood pressure is measured invasively in the operating room or intensive care unit, it is reasonable to expect useful systolic, diastolic, and mean pressure values to be displayed at all times. If blood pressure is stable from beat to beat, display of values from any beat will suffice. However, the pressure often fluctuates from beat to beat. Display of values from every beat would yield a confusing jumble of numbers. This article describes an algorithm for selecting values that appear physiological useful from blood pressure waveforms that vary with ventilation. Two classes of variation are discussed. The algorithm is optimized from the type of variation seen in pulmonary artery pressure waveforms.

Reorienting and expanding the physical map of temperate Bacillus subtilis bacteriophage phi 105.

During the course of extending the physical map of Bacillus subtilis temperate bacteriophage phi 105 to include SstI, XhoI, and HpaI, we recognized that the previous physical map for EcoRI was incorrect. The new enzyme maps were determined by single, double, and partial enzyme digestions, redigestion of purified phage fragments, end joint analysis, and DNA-DNA hybridization. The EcoRI physical map was corrected by double digestion of isolated fragments, DNA hybridization, and physical mapping by partial digestion of end-labeled fragments. EcoRI fragment G was repositioned to give the order D-G-I-E-B-H-F-C.

The effect of repeated administration of diftalone on enzyme induction.

Plasma concentrations of diftalone have been examined in normal volunteers after a single dose (500 mg) and after 500 mg doses given twice daily for one week. An increase in post dosing urinary excretion of D-glucaric acid showed a correlation with the ratio of calculated to observed areas under the plasma concentration, time curve following the final dose in the multiple dosing studies, indicating that hepatic microsomal enzymes are induced after repeated administration of the drug. Single dose studies in the presence of aluminium hydroxide and sodium bicarbonate showed that the antacids had no significant effect on the absorption of diftalone.