A Mutagenic Study of Functional and Structural Aspects of Rat Insulin-Like Growth Factor Binding Protein-5

On going research in our laboratory is focussed on the potential role of Insulin-like growth factor binding protein-5 (IGFBP-5) in apoptosis of mammary epithelial cells. In order to increase our understanding of IGFBP-5 functions, the work described below focussed on the relationship between the structure of the binding protein and its important molecular interactions. Using site-directed mutagenesis, we have mutated two highly conserved amino acids, Gly203 and Gln209 (G203K and Q209A respectively) within the basic amino acid rich region (201- 218) in the C-terminal domain of rat IGFBP-5. After analysis of binding activity using three different methods - IGF ligand blotting, IGF solution phase equilibrium binding and biosensor measurement - we have shown that the mutation of either of these two residues results in a reduction in affinity of the binding protein for both IGF-I and IGF-II by approximately ten-fold. Furthermore, mutation of both amino acids (G203K/Q209A, termed the Double mutant) had a cumulative effect and results in the complete ablation of IGF-I binding and a several thousand-fold reduction in IGF-II binding. This reduction in IGF binding affinity was comparable to that observed for the C-terminally truncated mutant BP550 (residues 1-168), which suggests that Gly203 and Gln209 may be the major determinants for IGF binding in the C-terminal domain. In addition, using heparin ligand blots, we confirm that mutation of basic residues within the C-terminal 201- 218 region (Hep- mutant: R201L, K202E, K206Q and R214A) results in major attenuation of heparin binding, whereas the G203K and Q209A single mutants and the Double mutant have no reduction in heparin binding. Therefore, our data suggests a potential overlap of heparin- and IGF- binding domains in the C-terminal region of IGFBP-5, and based on this, we discuss a potential model to explain the observed lower IGF binding affinity of ECM-bound IGFBP-5. In an attempt to assess the separate contribution of the N- and C-terminal domains to IGF binding, we also made four chimeric IGFBP cDNAs, BP552, BP522, BP255 and BP225, by switching domains between rat IGFBP-5 and rat IGFBP-2. We were consistently unable to detect expression of BP225 protein in the baculovirus/insect cell system, so this chimera had to be excluded from further study. Recognition of BP552 and BP522 proteins by both anti-rat IGFBP-5 and anti-rat IGFBP-2 antisera confirms their identity as chimeric proteins made up of domains from the native binding proteins, while BP255 was only recognised by the anti-rat IGFBP-5. The IGF binding properties of unpurified BP552, BP522 and BP255 proteins were assessed by IGF ligand blotting and IGF solution phase binding assays. These experiments demonstrated that BP552 and BP522 had comparable affinities with native IGFBP, whereas we were unable to detect any binding of BP255 to IGF-I by either technique, and only very weak interaction with IGF-II. Purified BP552 and BP522 were also tested by biosensor analysis, and data confirmed the results from the previous experiments. From this we can conclude that the N-terminal domain of IGFBP-5 can co-operate with the C-terminal domain of IGFBP-2 to produce a high affinity IGF binding species, irrespective of which central domain is present, whereas preliminary evidence would suggest that this cooperation may not occur between the N-terminal domain of IGFBP-2 and the C-terminal domain of lGFBP-5. In addition to IGF binding, the IGFBP-5 protein contains consensus heparin binding motifs in both its C-terminal and central domains, although only the C-terminal site was previously shown to be functional. Using heparin ligand blotting, it was found that the ability of BP552 and BP550 to bind to heparin was equivalent to that of WTIGFBP-5, whereas WTIGFBP-2 and BP522 failed to bind. These results demonstrate that an active heparin binding site in BP552 and BP550 is contained within the central domain of IGFBP-5, and that this site is only active in the absence of the carboxy-terminal domain. We subsequently mutated two basic amino acids (RI36A:R137A) in the central consensus binding sites between residues 132-140. This resulted in the loss of heparin binding for BP550, confirming the importance of these two basic amino acids in the central domain heparin binding activity. In light of these findings, we suggest that C-terminally truncated fragments of IGFBP-5 generated in vivo by proteolysis could retain heparin/extracellular matrix (ECM) binding properties. Finally, we propose that our various IGFBP-5 mutants described above provide an opportunity to test the structure-function relationships of this binding protein in an appropriate biological context

In Search of the Performer’s Way: The Work of Jerzy Grotowski from the Perspective of Daoism

The work of Jerzy Grotowski is investigated within the perspective of Daoism as it is discoursed in the classical Chinese text, Dao De Jing. In identifying its connection with the intellectual achievements of Grotowski’s contemporary Europe, i.e., Niels Bohr’s principle of complementarity in quantum physics and Jacques Derrida’s notion of différance in deconstructionism, Daoism provides an illuminating framework for comprehending Grotowski’s praxis. The philosophical notions of Daoism may be seen as underpinning Grotowski’s two most important principles, conjunctio oppositorum (conjunction of opposites) and via negativa (way of negation), whose implication can be encapsulated as the interaction of opposite pairs in the unceasing process. With these principles, the actors of the Theatre Laboratory accomplished the ‘total act’, an act of the body-text that embraces both one’s self and one’s cultural heritage, in the Poor Theatre in which novel actor/spectator relationships were also tested. In his post-theatrical work, Grotowski deepened his investigations of the performer’s body, which was finally identified as the body of essence of ‘the doer’. Grotowski’s lifetime research, in short, involved a persistent process of searching for the genuine body of the performer, which is clearly comprehended in the Daoist perspective that the world exists in the constant process of interplay between the fundamental pair of opposites, being (有, yǒu) and nonbeing (無, wú)

Short-Term Effects of Combined Serial Casting and Botulinum Toxin Injection for Spastic Equinus in Ambulatory Children with Cerebral Palsy

PURPOSE: The purpose of this paper is to test the hypothesis that combination therapy of serial cast and botulinum toxin type A (BTX-A) injection can further enhance the effects of a BTX-A injection in ambulant children with cerebral palsy (CP) who have an equinus foot. MATERIALS AND METHODS: Children in group A (30 legs of 21 children) received a serial casting application after an injection of BTX-A, and children in group B (25 legs of 17 children) received only a BTX-A injection. Assessments were performed before the intervention and 1 month after the intervention. RESULTS: After the intervention, there were significant improvements in tone, dynamic spasticity, and passive range of motion (ROM) in both groups. However, the changes were greater in group A than in group B. Dimension D (standing) in Gross Motor Function Measure (GMFM)-66 was significantly improved in group A but not in group B. On the other hand, there were no significant changes in dimension E (walking, running, jumping) in GMFM-66 in either group. CONCLUSION: The results of our study suggest that a serial casting application after BTX-A injection can enhance the benefits of BTX-A injection in children with cerebral palsy.ope

Intraoperative blood loss during different stages of scoliosis surgery: A prospective study

<p>Abstract</p> <p>Background</p> <p>There are a number of reasons for intraoperative blood loss during scoliosis surgery based on the type of approach, type of disease, osteopenia, and patient blood profile. However, no studies have investigated bleeding patterns according to the stage of the operation. The objective of this prospective study was to identify intraoperative bleeding patterns in different stages of scoliosis surgery.</p> <p>Methods</p> <p>We prospectively analyzed the estimated blood loss (EBL) and operation time over four stages of scoliosis surgery in 44 patients. The patients were divided into three groups: adolescent idiopathic (group 1), spastic neuromuscular (group 2) and paralytic neuromuscular (group 3). The per-level EBL and operation times of the groups were compared on a stage-by-stage basis. The bone marrow density (BMD) of each patient was also obtained, and the relationship between per-level EBL and BMD was compared using regression analysis.</p> <p>Results</p> <p>Per-level operation time was similar across all groups during surgical stage (p > 0.05). Per-level EBL was also similar during the dissection and bone-grafting states (p > 0.05). However, during the screw insertion stage, the per-level EBL was significantly higher in groups 2 and 3 compared to group 1 (p < 0.05). In the correction stage, per-level EBL was highest in group 3 (followed in order by groups 2 and 1) (p < 0.05). Preoperative BMD indicated that group 3 had the lowest bone quality, followed by groups 2 and 1 (in order), but the preoperative blood indices were similar in all groups. The differences in bleeding patterns in the screw insertion and correction stages were attributed to the poor bone quality of groups 2 and 3. Group 3 had the lowest bone quality, which caused loosening of the bone-screw interface during the correction stage and led to more bleeding. Patients with a T-score less than -2.5 showed a risk for high per-level EBL that was nine times higher than those with scores greater than -2.5 (p = 0.003).</p> <p>Conclusions</p> <p>We investigated the blood loss patterns during different stages of scoliosis surgery. Patients with poor BMD showed a risk of blood loss nine times higher than those with good BMD.</p

DS-ARP: A New Detection Scheme for ARP Spoofing Attacks Based on Routing Trace for Ubiquitous Environments

Despite the convenience, ubiquitous computing suffers from many threats and security risks. Security considerations in the ubiquitous network are required to create enriched and more secure ubiquitous environments. The address resolution protocol (ARP) is a protocol used to identify the IP address and the physical address of the associated network card. ARP is designed to work without problems in general environments. However, since it does not include security measures against malicious attacks, in its design, an attacker can impersonate another host using ARP spoofing or access important information. In this paper, we propose a new detection scheme for ARP spoofing attacks using a routing trace, which can be used to protect the internal network. Tracing routing can find the change of network movement path. The proposed scheme provides high constancy and compatibility because it does not alter the ARP protocol. In addition, it is simple and stable, as it does not use a complex algorithm or impose extra load on the computer system