ADAMTS-13 glycans and conformation-dependent activity.

Essentials The impact of N-linked glycosylation on ADAMTS-13 function has not been fully explored. The activity of glycan modified ADAMTS-13 was investigated under static and shear stress conditions. Terminal sialic acid on the metalloprotease domain glycans are important for ADAMTS-13 activity. The CUB domain glycans modulate ADAMTS-13 activity. SUMMARY: Background ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation. Objectives/Methods We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions. Results Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain. Conclusions Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.

Case Study: Supplementation of cow-calf pairs grazing smooth bromegrass pastures with ethanol by-products and low-quality forages.

Multiparous, lactating, crossbred (Simmental × Angus) beef cows with spring-born calves at side (n = 16 per year; 4 per pasture) were used each of 3 yr to evaluate supplementing modified distillers grains plus solubles mixed with low-quality forage on cow and calf performance while grazing. Cow-calf pairs were assigned randomly to treatment with 2 replications (pasture) per year for 3 yr. Treatments were (1) recommended stocking rate of 9.46 animal-unit month/ha with no supplementation (CON) or (2) double the recommended stocking rate (18.9 animal-unit month/ha) and supplemented with a 30:70 modified distillers grains plus solubles:cornstalks (DM) mixture (SUPP). To replace 50% of grazed forage DMI, SUPP pairs were fed an average of 1.13% of BW (DM) over the grazing season. Pairs grazed adjacent smooth bromegrass pastures for 130 d during the summer. Gain was not different (P = 0.19) between SUPP and CON cows (0.28 vs. 0.19 kg/d, respectively). Ending cow BW was not affected (P = 0.46) by treatment. Similarly, calf gain was not affected (P = 0.31) by supplementation. In studies where confined cow-calf pairs were fed average-quality (IVDMD = 52.9%) forage, DMI was 2.58% of pair BW. Based on these data, CON and SUPP pairs consumed 18.6 and 19.1 kg of DM, respectively, of total feed per pair daily. The SUPP pairs consumed 7.1 kg of DM/pair daily of the supplement, replacing approximately 35% of grazed forage intake. These data suggest mixtures of ethanol co-products and low-quality forages can be supplemented to replace grazed forage intake of cattle, allowing for increased stocking rate without affecting animal performance.

Surface freezing in binary mixtures of chain molecules. II. Dry and hydrated alcohol mixtures.

Surface freezing is studied in dry and hydrated alcohol mixtures by surface x-ray scattering and surface tension measurements. A crystalline bilayer is formed at the surface a few degrees above the bulk freezing temperature. The packing is hexagonal, with molecules aligned along the surface normal in all cases. The in-plane lattice constant reveals a qualitatively different behavior with composition for hydrated and dry mixtures. The simple theoretical approach used successfully for alkane and deuterated alkane mixtures accounts well also for the alcohol mixtures. The repulsive length-mismatch term opposing the mixing entropy term in the free energy of the mixtures is shown to have a universal behavior for all mixtures studied: protonated alkanes, deuterated alkanes, and dry and wet alcohols. This universality is somewhat counterintuitive in view of the different interactions (e.g., hydrogen bonding in alcohols) in the different mixtures.

Preservation of ejaculation through a modified retroperitoneal lymph node dissection in low stage testis cancer.

We studied ejaculatory function in 47 patients who underwent modified retroperitoneal lymph node dissection for nonseminomatous germ cell tumors of the testis from 1983 to 1989. Our goal was to assess the effectiveness of a modified node dissection in preserving ejaculatory function while eliminating the risk of retroperitoneal recurrence. There were 13 left and 34 right tumors. Of the patients 45 had clinical stage A cancer, and 2 had clinical stage B2 disease and received preoperative chemotherapy. The template method of dissection was used, which spares the preaortic area below the inferior mesenteric artery, including the sacral promontory. Average operating time was 3 hours 40 minutes. There was no operative mortality. Spontaneous recovery of ejaculation occurred in 38 patients (81%), more commonly with right (88%) than with left dissections (62%). The mean interval to recovery of ejaculation was 5 months (92% within 12 months). Five patients (10%) had recurrence, with a mean followup of 28 months. All recurrences were in the lungs 4 to 8 months after retroperitoneal lymph node dissection, and all patients were salvaged with chemotherapy and are disease-free. We confirmed that a modified template-type retroperitoneal lymph node dissection can be accomplished safely with preservation of ejaculation in more than 80% of the patients without increasing the risk of local recurrence.

A complication of interference screw fixation in anterior cruciate ligament reconstruction.

Bone-tendon autografts using patellar tendon are rapidly becoming the autogenous grafts of choice for anterior cruciate ligament reconstruction. Patellar tendon is the strongest type of available autogenous tissue, and it is easily adaptable to arthroscopically assisted techniques. Secure immediate fixation is critical for the success of this procedure. The Kurosaka screw has emerged as one of the most reliable graft fixation methods. Although we have enjoyed much success with the Kurosaka screw, the following case represents the first report of failure with this fixation technique.

Ionic currents contributing to the action potential in single ventricular myocytes of the guinea pig studied with action potential clamp.

With the action potential clamp procedure we studied the contribution of various ionic currents to the action potential in single ventricular myocytes. Action potentials were elicited by a current pulse through the suction pipette and recorded by a computer. A representative action potential was then repetitively replayed to the same cell under voltage-clamp conditions. Successive pharmacological blocks of ionic currents allowed for the first time the measurement of the contribution of the L-type calcium current (ICa) and the [Ca2+]i-activated currents as well as the potassium current to the action potential. Experiments using caffeine as a tool to increase calcium release from the sarcoplasmic reticulum supported the idea that INaCa contributes to the plateau during the second half of the action potential and even lasts into diastole, whereas strong elevation of the intracellular [Ca]i during the action potential additionally activated the non-specific cation channel.

[Leu5]enkephalin inhibits norepinephrine-induced contraction of rat aorta.

Leucine-enkephalin produces a dose related decrease in the contractile response of spirally cut strips of rat aorta to norepinephrine (NE). The maximal effect (55.0 +/- 1.0 percent reduction in developed tension) occurs at a leucine-enkephalin concentration of 10(-10) M. The attenuation of contractile response is reversed by naloxone.