Effect of disulfide bridge formation on the NMR spectrum of a protein: Studies on oxidized and reduced Escherichia coli thioredoxin

Summary As a prelude to complete structure calculations of both the oxidized and reduced forms of Escherchia coli thioredoxin (M_r 11 700), we have analyzed the NMR data obtained for the two proteins under identical conditions. The complete aliphatic ¹³C assignments for both oxidized and reduced thioredoxin are reported. Correlations previously noted between ¹³C chemical shifts and secondary structure are confirmed in this work, and significant differences are observed in the C and C shifts between cis- and trans-proline, consistent with previous work that identifies this as a simple and unambiguous method of identifying cis-proline residues in proteins. Reduction of the disulfide bond in the active-site Cys³²-Gly-Pro-Cys³⁵ sequence causes changes in the ¹H, ¹⁵N and ¹³C chemical shifts of residues close to the active site, some of them quite far distant in the amino acid sequence. Coupling constants, both backbone and side chain, show some differences between the two proteins, and the NOE connectivities and chemical shifts are consistent with small changes in the positions of several side chains, including the two tryptophan rings (Trp²⁸ and Trp³¹). These results show that, consistent with the biochemical behavior of thioredoxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein.     

A limited-trust capacity model for mitigating threats of internal malicious services in cloud computing

Hidden persistent malware in guest virtual machine instances are among the most common internal threats in cloud computing, affecting the security of both cloud customers and providers. With the growing sophistication of modern malware, traditional methods are becoming increasingly ineffective for tackling cloud security problems. Moreover, given the pay-per-use model of clouds, consumption of resources by these malwares and malicious services can cause huge losses to both the cloud provider and customer. Thus, it is important to develop mechanisms that can limit the scale of malicious attacks in order to minimize their resources consumption. Trust management is a fundamental technique for assessing and increasing the reliability and security of cloud services. Unfortunately, majority of existing mechanisms for trust management in clouds have limitations that prevent them from being fully effective. In this paper, we propose a novel limited-trust capacity model to mitigate the threats of internal malicious software and services in cloud computing using concepts from flow networks to reduce the scale of malicious software or services. Our limited-trust capacity model can be utilized in the following two ways: (1) to manage the trust relationship among the guest services and to evaluate the threats of unknown malicious services, and (2) to minimize risk associated with renting cloud services and limiting the resource drain caused by malicious guest services. Finally, experimental results show that our limited-trust capacity model can effectively restrict the scale of malicious services and significantly mitigate the threats of internal attacks.     

Characterization of antiviral and antibacterial activity of Bombyx mori seroin proteins

Lepidopterans as other insects have a very potent innate immune system, which basically comprises cellular and humoral defence mechanisms against bacterial and fungal infections. In lepidopterans, not much is known about the defence mechanisms against viral pathogens, such as baculoviruses. Here we show that small silk proteins of the domesticated silkworm, Bombyx mori, called seroins, act as antiviral agents against a baculovirus pathogen, Bombyx mori nucleopolyhedrovirus (BmNPV). Involvement of these proteins in the inhibition of baculovirus infection was revealed by estimating the viral load upon their dsRNA‐mediated knockdown. Additionally, we found through antimicrobial assays that seroins are potent inhibitors of bacterial growth. Binding competition assays followed by antimicrobial assays showed that seroins bind to peptidoglycan, a cell wall component of bacteria. Analysis of bacterial load upon knockdown of seroins resulted in higher proliferation of bacteria. Phylogenetic analysis showed the recent origin of seroins in a few moth species and duplication only in Bombycids. The antiviral and antibacterial activity of seroins shown in this study using several biochemical and molecular biological assays provide strong evidence to characterize them as antimicrobial proteins. Hence, we hypothesize that seroins are potent candidates for use in development of transgene‐based disease resistant silkworm strains.     

Trunk, abdomen, and pressure sore reconstruction

LEARNING OBJECTIVES: After reading this article, the participant should be able to: 1. Describe the principles of wound closure, torso reconstruction, and pressure sore reconstruction. 2. Outline standard options to treat defects of the chest, abdomen, and back and pressure ulcers in all anatomical areas. 3. Manage and prevent pressure ulcers. SUMMARY: Chest wall reconstruction is indicated following tumor resection, radiation wound breakdown, or intrathoracic sepsis. Principles of wound closure and chest wall stabilization, where indicated, are discussed. Principles of abdominal wall reconstruction continue to evolve with the introduction of newer bioprosthetics and the application of functional concepts for wound closure. The authors illustrate these principles using commonly encountered clinical scenarios and guidelines to achieve predictable results. Pressure ulcers continue to be devastating complications to patients' health and a functional hazard when they occur in the bedridden, in patients with spinal cord injuries, and in patients with neuromuscular disease. Management of pressure ulcers is also very expensive. The authors describe standard options to treat defects of the chest, abdomen, and back and pressure ulcers in all anatomical areas. A comprehensive understanding of principles and techniques will allow practitioners to approach difficult issues of torso reconstruction and pressure sores with a rational confidence and an expectation of generally satisfactory outcomes. With pressure ulcers, prevention remains the primary goal. Patient education and compliance coupled with a multidisciplinary team approach can reduce their occurrence significantly. Surgical management includes appropriate patient selection, adequate débridement, soft-tissue coverage, and use of flaps that will not limit future reconstructions if needed. Postoperatively, a strict protocol should be adapted to ensure the success of the flap procedure. Several myocutaneous flaps commonly used for the surgical management of pressure are discussed. Commonly used flaps in chest and abdominal wall reconstruction are discussed and these should be useful for the practicing plastic surgeon.     

Crystal structure of the C2 domain of class II phosphatidylinositide 3-kinase C2alpha

Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3'-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel beta-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) and that three lysine residues at SBS I site, Lys-1420, Lys-1432, and Lys-1434, are responsible for the phospholipid binding affinity.     

NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair

In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA–XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER.