Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (α , β , and γ ), and also their two-domain combinations, viz. αβ and βγ in Escherichia coli. After purification, α and β showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs αβ and βγ showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK·HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK·HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels

Involvement of a nine-residue loop of streptokinase in the generation of macromolecular substrate specificity by the activator complex through interaction with substrate kringle domains

The selective deletion of a discrete surface-exposed epitope (residues 254-262; 250-loop) in the β domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SKdel254-262). A kinetic analysis of SKdel254-262 revealed that its low HPG activator activity arose from a 5-6-fold increase in Km for HPG as substrate, with little alteration in kcat rates. This increase in the Km for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SKdel254-262. The involvement of kringles was further indicated by a hypersusceptibility of the SKdel254-262. plasmin activator complex to ε-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK.plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SKdel254-262.HPG was reduced by about 3-fold in comparison with that of nSK.HPG. Overall, these observations identify the 250 loop in the β domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK.HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process

Review: Endoscopic ultrasound-guided fine needle injection for cancer therapy: The evolving role of therapeutic endoscopic ultrasound

Endoscopic ultrasound (EUS) is central to the diagnosis and staging of many malignancies, but now has an evolving role in cancer therapy. EUS-guided fine needle injection (FNI) is already used for palliative interventions such as treatment of pain through nerve blockade and to guide biliary decompression when conventional ERCP is not possible. More recently, EUS-FNI has been used to deliver specific anti-tumor agents for pancreatic cyst ablation and local control of tumor growth in patients with unresectable solid malignancies. The agents used to date include ethanol, brachytherapy seeds, and chemotherapeutic agents such as paclitaxel. In addition, FNI of new immunomodulating cell cultures such as mixed lymphocyte and dendritic cell cultures has also been reported, as has FNI of several different viral vectors for antitumor therapy. Although experience with these agents remains preliminary, EUS-FNI is a minimally invasive approach to deliver local antitumor agents, and is likely to have an expanding role in cancer therapy

Is there any difference in outcomes between long pigtail and short flanged prophylactic pancreatic duct stents?

OBJECTIVE: Prophylactic pancreatic duct (PD) stent placement has been shown to reduce the incidence of post-ERCP pancreatitis (PEP) especially in high-risk patients. However, there is no consensus on the best type of PD stent. The purpose of our study was to evaluate the differences in the outcomes between long (\u3e3 cm) pigtail and short (≤3 cm) flanged 4 Fr Freeman Pancreatic Flexi-Stents in preventing PEP. METHODS: We retrospectively reviewed all ERCP procedures performed between 08/01/2006 and 10/01/2007 by one of two experienced endoscopists (\u3e5 years of experience) with the assistance of a trainee. Patient data was collected for indications, risk factors for PEP, type and reason for PD stent, complications, and any mortality. The PD stent was removed endoscopically if it was still in place on abdominal X-ray done 2 weeks post-ERCP. The data was analyzed with Student\u27s t test, Chi-square, and ANOVA tests by using SPSS software version 15.0. RESULTS: Out of a total of 753 ERCP procedures, 179 (23.8%) required either long or short prophylactic PD stents. The incidence of PEP was 3.7% versus 13.6% for long and short stent groups, respectively (p=0.019). Spontaneous stent dislodgement rate was 95.4% versus 81.8% for long and short stent groups, respectively (p=0.007). There was no difference in non-pancreatic complications between the two stent groups. There was no procedure-related mortality. CONCLUSIONS: Long (\u3e3 cm) pigtail PD stent due to their specific design showed better outcomes as compared to short (\u3c3 \u3ecm) flanged PD stent in preventing PEP and spontaneous stent dislodgement rates. However, further prospective trials are needed