Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Long-Term Clinical Outcome of Internal Globus Pallidus Deep Brain Stimulation for Dystonia

Background

GPi (Internal globus pallidus) DBS (deep brain stimulation) is recognized as a safe, reliable, reversible and adjustable treatment in patients with medically refractory dystonia.

Objectives

This report describes the long-term clinical outcome of 36 patients implanted with GPi DBS at the Neurosurgery Department of Seoul National University Hospital.

Methods

Nine patients with a known genetic cause, 12 patients with acquired dystonia, and 15 patients with isolated dystonia without a known genetic cause were included. When categorized by phenomenology, 29 patients had generalized, 5 patients had segmental, and 2 patients had multifocal dystonia. Patients were assessed preoperatively and at defined follow-up examinations postoperatively, using the Burke-Fahn-Marsden dystonia rating scale (BFMDRS) for movement and functional disability assessment. The mean follow-up duration was 47 months (range, 12–84)

Results

The mean movement scores significantly decreased from 44.88 points preoperatively to 26.45 points at 60-month follow up (N = 19, P = 0.006). The mean disability score was also decreased over time, from 11.54 points preoperatively to 8.26 points at 60-month follow up, despite no statistical significance (N = 19, P = 0.073). When analyzed the movement and disability improvement rates at 12-month follow up point, no significant difference was noted according to etiology, disease duration, age at surgery, age of onset, and phenomenology. However, the patients with DYT-1 dystonia and isolated dystonia without a known genetic cause showed marked improvement.

Conclusions

GPi DBS is a safe and efficient therapeutic method for treatment of dystonia patients to improve both movement and disability. However, this study has some limitations caused by the retrospective design with small sample size in a single-center.     

Mutagenicity and polycyclic aromatic hydrocarbon content of fumes from heated cooking oils produced in Taiwan

According to epidemiologic studies, exposure of women to fumes from cooking oils appears to be an important risk factor for lung cancer. Fume samples from three different commercial cooking oils frequently used in Taiwan were collected and analyzed for mutagenicity in the Salmonella/microsome assay. Polycyclic aromatic hydrocarbons were extracted from the samples and identified by HPLC chromatography. Extracts from three cooking oil fumes were found to be mutagenic in the presence of S9 mix. All samples contained dibenz[a,h]anthracene (DB[a,h]A) and benz[a]anthracene (B[a]A). Concentration of DB[a,h]A and B[a]A were 1.9 and 2.2 μg/m³ in fumes from lard oil, 2.1 and 2.3 μg/m³ in soybean oil, 1.8 and 1.3 μg/m³ in peanut oil, respectively. Benzo[a]pyrene (B[a]P) was identified in fume samples of soybean and peanut oil, in concentrations of 19.6 and 18.3 μg/m³, in this order. These results provide experimental evidence and support the findings of epidemiologic observations, in which women exposed to the emitted fumes of cooking oils are at increased risk of contracting lung cancer.     

The MHC class I-like IgG receptor controls perinatal IgG transport,IgG homeostasis,and fate of IgG-Fc-coupled drugs

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.     

A direct test of the reductionist approach to structural studies of calmodulin activity: relevance of peptide models of target proteins

Ca(2+)-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain. Using heteronuclear NMR (13)C-(15)N-(1)H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI. A comparison of chemical shifts for free CaM with those of the CaM.CaMKIp complex indicate large differences throughout the CaM sequence. Using NMR techniques optimized for large proteins, backbone resonance assignments were also determined for CaM bound to the intact CaMKI enzyme. NMR spectra of CaM bound to either the CaMKI enzyme or peptide are virtually identical, indicating that calmodulin is structurally indistinguishable when complexed to the intact kinase or the peptide CaM-binding domain. Chemical shifts of CaM bound to a peptide (smMLCKp) corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are also compared with the CaM.CaMKI complexes. Chemical shifts can differentiate one complex from another, as well as bound versus free states of CaM. In this context, the observed similarity between CaM.CaMKI enzyme and peptide complexes is striking, indicating that the peptide is an excellent mimetic for interaction of calmodulin with the CaMKI enzyme.     

Southern Ocean Biogeography of Tintinnid Ciliates of the Marine Plankton

Ciliate microzooplankton are important grazers in most pelagic ecosystems and among them, tintinnids, with their largely species‐specific loricas, allow relatively easy assessment of questions of diversity and distributions. Herein, we present the results of a survey of species records of tintinnids from the Southern Ocean (locations below 40°S) reported in 56 publications yielding 2,047 species records (synonyms included) from 402 locations. The 192 species reported can be parsed into two main groups: 32 endemic Southern Ocean species, known only from 40°S and further south, and a second group of 181 widespread species, forms with extensive geographic ranges extending into the Southern Ocean. Widespread species reported from the Southern Ocean can be further divided into a group of 81 species, each recorded multiple times in the Southern Ocean waters and 70 apparent “stray” species which have only been found but once. The endemic and widespread species of the Southern Ocean show both distinct distributional patterns and morphological differences. The assemblage of Southern Ocean endemics is found mostly within the Antarctic zone delimited by the average location of the Polar Front and contains a relatively large portion of wide‐mouthed forms. We give suggestions for future study.     

Biochemical analysis of a Chinese cabbage phytocystatin-1

The phytocystatins are inhibitors of papain-like cysteine proteinases that are implicated in defense mechanisms and the regulation of protein turnover. BCPI-1, a Brassica rapa (Chinese cabbage) phytocystatin isolated from flower buds, contains an extended C-terminal region that contains a single Cys residue at position 102. In an effort to investigate the role of the C-terminus and this Cys residue in BCPI-1 activity, purified recombinant proteins of BCPI-1, including wild-type BCPI-1 (wtBCPI-1), N-terminus BCPI-1 (BCPI-1??C), C-terminus BCPI-1 (BCPI-1??N), and BCPI-1 with a single Cys residue exchange to Ser (BCPI-1C102S), were generated and their inhibitory activities against papain were investigated. Kinetic analysis revealed that the monomeric forms of wtBCPI-1 (K _i = 6.84 ± 0.3 × 10^?8 M) inhibited papain more efficiently than the dimeric forms of wtBCPI-1 (K _i = 1.01 ± 0.5 × 10^?7 M). Experiments with recombinant BCPI-1C102S demonstrated that the dimerization of wtBCPI-1 caused by the formation of an intermolecular disulfide bond at the cysteine residue. The inhibitory activity of the recombinant proteins, except BCPI-1??N, was reduced in the pH range of 7.0?C11.5 and was highly stable over a wide range of temperatures. Thus, dimerization mediated by the cysteine residue in the extended C-terminal region and alkaline conditions reduced the inhibitory activity of BCPI-1.     

Inverse Association between Glycated Albumin and Insulin Secretory Function May Explain Higher Levels of Glycated Albumin in Subjects with Longer Duration of Diabetes

Background

Glycated albumin (GA) has been increasingly used as a reliable index for short-term glycemic monitoring, and is inversely associated with β-cell function. Because the pathophysiologic nature of type 2 diabetes (T2D) is characterized by progressive decline in insulin secretion, the aim was to determine whether GA levels were affected by diabetes duration in subjects with T2D.

Methods

To minimize the effect of glucose variability on GA, subjects with stably maintained HbA_1c levels of <0.5% fluctuation across 6 months of measurements were included. Patients with newly diagnosed T2D (n = 1059) and with duration>1 year (n = 781) were recruited and categorized as New-T2D and Old-T2D, respectively. Biochemical, glycemic, and C-peptide parameters were measured.

Results

GA levels were significantly elevated in HbA_1c-matched Old-T2D subjects compared to New-T2D subjects. Duration of diabetes was positively correlated with GA, whereas a negative relationship was found with C-peptide increment (ΔC-peptide). Among insulin secretory indices, dynamic parameters such as ΔC-peptide were inversely related to GA (r = −0.42, p<0.001). Multiple linear regression analyses showed that duration of diabetes was associated with GA (standardized β coefficient [STDβ] = 0.05, p<0.001), but not with HbA_1c (STDβ = 0.04, p<0.095). This association disappeared after additional adjustment with ΔC-peptide (STDβ = 0.02, p = 0.372), suggesting that β-cell function might be a linking factor of close relationship between duration of diabetes and GA values.

Conclusions

The present study showed that GA levels were significantly increased in subjects with longer duration T2D and with decreased insulin secretory function. Additional caution should be taken when interpreting GA values to assess glycemic control status in these individuals.