Claudin-4 is required for AMPK-modulated paraceliular permeability in submandibular gland ceils

Tight junction plays an important rote in mediating paraceUular permeability in epithelia. We previously found that activation of AMP- activated protein kinase （AMPK） increased saliva secretion by modulating paraceUular permeability in submandibular glands. However, the molecular mechanisms underlying AMPK-modulated paraceUular permeability are unknown. In this study, we found that AICAR, an AMPK agonist, increased saliva secretion in the isolated rat submandibular glands, decreased transepithelial electrical resistance （TER）, and increased 4 kDa FITC-dextran flux in cultured SMG-C6 cells. AICAR also induced redistribution of tight junction protein claudin-4, but not claudin-1, claudin-3, occtudin, or ZO-1, from the cytoplasm to the membrane. Moreover, knockdown of claudin-4 by shRNA suppressed while claudin-4 re-expression restored the TER and 4 kDa FITC-dextran flux responses to AICAR. Additionally, AICAR increased ERK1/2 phosphorylation, and inhibition of ERK1/2 by U0126, an ERK1/2 kinase inhibitor, or by siRNA decreased AICAR-induced TER responses. AICAR induced the serine S199 phosphorylation of claudin-4 and enhanced the inter- action of claudin-4 and occludin. Furthermore, pretreatment with U0126 significantly suppressed AMPK-modulated phosphorytation, redistribution, and interaction with occludin of claudin-4. Taken together, these results indicated that claudin-4 played a crucial role in AMPK-rnodutated paraceUular permeability and ERK1/2 was required in AMPK-modulated tight junction barrier function in subman- dibular gland.     

IL-31 Associated with Coronary Artery Lesion Formation in Kawasaki Disease

Background

Kawasaki disease (KD) is known to be associated with T help (Th) 2 reaction and subsequently allergic diseases. Interleukin-31 (IL-31) has also been reported to be involved in Th2 mediated diseases such as allergic diseases. However, the role of IL-31 in KD has not been previously reported. The aim of this study is to investigate whether IL-31 is associated with KD and its clinical outcome.

Material

A total of 78 KD patients who met the criteria of KD were enrolled in this study as well as 20 age-matched controls. Plasma samples were conducted to measure IL-31 before intravenous immunoglobulin (IVIG) treatment (KD1), within 3 days after IVIG treatment (KD2) and at least 3 weeks after IVIG treatment (KD3) by utilizing enzyme-linked immunosorbent assay (ELISA).

Result

Our findings showed that IL-31 expression was higher in KD patients after IVIG treatment significantly (KD2>KD1: 1265.0±199.3 vs. 840.2±152.5 pg/ml, p<0.0001). Further analysis revealed that IL-31 level was significantly higher in KD patients with coronary artery lesion (CAL) (656.6±139.5 vs. 1373.0±422.0 pg/ml, p = 0.04) before IVIG treatment (KD1). There were no significant differences between the IVIG resistance and IVIG responsiveness groups.

Conclusion

IL-31 was increased after IVIG treatment in patients with KD and was significantly associated with CAL formation. The results from this study may help to identify a novel risk factor for predicting KD and CAL formation.     

Laser Acupuncture Therapy in Patients with Treatment-Resistant Temporomandibular Disorders

Objective

To investigate the clinical effects of laser acupuncture therapy for temporomandibular disorders (TMD) after ineffective previous treatments.

Methods

A retrospective observational study was conducted in 29 treatment-resistant TMD patients (25 women, 4 men; age range, 17–67 years). Subjects were treated 3 times per week for 4 weeks with the Handylaser Trion (GaAlAs laser diode, 810 nm, 150 mW, pulsed waves), which delivered 0.375 J of energy (5 s) to ST7, ST6, and LI4 and 3 J (40 s) to each Ashi point, 7.5–26.25 J/cm² in total. The visual analog scale (VAS) and maximal mouth opening (MMO) were evaluated before and after treatment.

Results

VAS analysis showed that the patients were free of pain at rest (endpoint) after 5.90±6.08 sessions of laser acupuncture for acute TMD and after 16.21±17.98 sessions for chronic TMD. The VAS score on palpation of the temporomandibular joint reduced to 0.30±0.67 for patients with acute TMD (p = 0.005) and to 0.47±0.84 for those with chronic TMD (p<0.001). The MMO significantly increased in patients with acute TMD (7.80±5.43 mm, p = 0.008) and in patients with chronic TMD (15.58±7.87 mm, p<0.001).

Conclusions

Our study shows that laser acupuncture therapy improves the symptoms of treatment-resistant TMD. Further studies with a more appropriate design, involving long-term follow-up examinations in a larger patient sample, are needed to evaluate its efficacy.     

Diverse Virulence Gene Content of Shiga Toxin-Producing Escherichia coli from Finishing Swine

Shiga toxin-producing Escherichia coli (STEC) infections are a critical public health concern because they can cause severe clinical outcomes, such as hemolytic uremic syndrome, in humans. Determining the presence or absence of virulence genes is essential in assessing the potential pathogenicity of STEC strains. Currently, there is limited information about the virulence genes carried by swine STEC strains; therefore, this study was conducted to examine the presence and absence of 69 virulence genes in STEC strains recovered previously from finishing swine in a longitudinal study. A subset of STEC strains was analyzed by pulsed-field gel electrophoresis (PFGE) to examine their genetic relatedness. Swine STEC strains (n = 150) were analyzed by the use of a high-throughput real-time PCR array system, which included 69 virulence gene targets. Three major pathotypes consisted of 16 different combinations of virulence gene profiles, and serotypes were determined in the swine STEC strains. The majority of the swine STEC strains (n = 120) belonged to serotype O59:H21 and carried the same virulence gene profile, which consisted of 9 virulence genes: stx_2e, iha, ecs1763, lpfA_O113, estIa (STa), ehaA, paa, terE, and ureD. The eae, nleF, and nleH1-2 genes were detected in one swine STEC strain (O49:H21). Other genes encoding adhesins, including iha, were identified (n = 149). The PFGE results demonstrated that swine STEC strains from pigs raised in the same finishing barn were closely related. Our results revealed diverse virulence gene contents among the members of the swine STEC population and enhance understanding of the dynamics of transmission of STEC strains among pigs housed in the same barn.     

Affinity purification of Candida albicans CaCdc4-associated proteins reveals the presence of novel proteins involved in morphogenesis

Candida albicans CDC4 is nonessential and plays a role in suppressing filamentous growth, in contrast to its evolutionary counterparts involved in the G1-S transition of the cell cycle. Genetic epistasis analysis has indicated that proteins besides Sol1 are targets of C. albicans Cdc4. Moreover, no formal evidence suggests that C. albicans Cdc4 functions through the ubiquitin E3 ligase of the Skp1-Cul1/Cdc53-F-box complex. To elucidate the role of C. albicans CDC4, C. albicans Cdc4-associated proteins were sought by affinity purification. A 6×His epitope-tagged C. albicans Cdc4 expressed from Escherichia coli was used in affinity purifications with the cell lysate of C. albicans cdc4 homozygous null mutant. Candida albicans Cdc4 and its associated proteins were resolved by SDS-PAGE and visualized by silver staining. The candidate proteins were recovered and trypsin-digested to generate MALDI-TOF spectra profiles, which were used to search against those of known proteins in the database to reveal their identities. Two out of four proteins encoded by GPH1 and THR1 genes were further verified to interact with C. albicans Cdc4 using a yeast two-hybrid assay. We conclude that in vitro affinity purification using C. albicans Cdc4 generated from E. coli as the bait and proteins from cell lysate of C. albicans cdc4 homozygous null mutant as a source of prey permit the identification of novel proteins that physically interact and functionally associate with C. albicans Cdc4.     

The S Helix Mediates Signal Transmission as a HAMP Domain Coiled-Coil Extension in the NarX Nitrate Sensor from Escherichia coli K-12

In the nitrate-responsive, homodimeric NarX sensor, two cytoplasmic membrane α-helices delimit the periplasmic ligand-binding domain. The HAMP domain, a four-helix parallel coiled-coil built from two α-helices (HD1 and HD2), immediately follows the second transmembrane helix. Previous computational studies identified a likely coiled-coil-forming α-helix, the signaling helix (S helix), in a range of signaling proteins, including eucaryal receptor guanylyl cyclases, but its function remains obscure. In NarX, the HAMP HD2 and S-helix regions overlap and apparently form a continuous coiled-coil marked by a heptad repeat stutter discontinuity at the distal boundary of HD2. Similar composite HD2-S-helix elements are present in other sensors, such as Sln1p from Saccharomyces cerevisiae. We constructed deletions and missense substitutions in the NarX S helix. Most caused constitutive signaling phenotypes. However, strongly impaired induction phenotypes were conferred by heptad deletions within the S-helix conserved core and also by deletions that remove the heptad stutter. The latter observation illuminates a key element of the dynamic bundle hypothesis for signaling across the heptad stutter adjacent to the HAMP domain in methyl-accepting chemotaxis proteins (Q. Zhou, P. Ames, and J. S. Parkinson, Mol. Microbiol. 73:801-814, 2009). Sequence comparisons identified other examples of heptad stutters between a HAMP domain and a contiguous coiled-coil-like heptad repeat sequence in conventional sensors, such as CpxA, EnvZ, PhoQ, and QseC; other S-helix-containing sensors, such as BarA and TorS; and the Neurospora crassa Nik-1 (Os-1) sensor that contains a tandem array of alternating HAMP and HAMP-like elements. Therefore, stutter elements may be broadly important for HAMP function.Transmembrane signaling in homodimeric bacterial sensors initiates upon signal ligand binding to the extracytoplasmic domain. In methyl-accepting chemotaxis proteins (MCPs), the resulting conformational change causes a displacement of one transmembrane α-helix (TM α-helix) relative to the other. This motion is conducted by the HAMP domain to control output domain activity (reviewed in references 33 and 39).Certain sensors of two-component regulatory systems share topological organization with MCPs. For example, the paralogous nitrate sensors NarX and NarQ contain an amino-terminal transmembrane signaling module similar to those in MCPs, in which a pair of TM α-helices delimit the periplasmic ligand-binding domain (Fig. ​(Fig.1)1) (24) (reviewed in references 32 and 62). The second TM α-helix connects to the HAMP domain. Hybrid proteins in which the NarX transmembrane signaling module regulates the kinase control modules of the MCPs Tar, DifA, and FrzCD demonstrate that NarX and MCPs share a mechanism for transmembrane signaling (73, 74, 81, 82).Open in a separate windowFIG. 1.NarX modular structure. Linear representation of the NarX protein sequence, from the amino (N) to carboxyl (C) termini, drawn to scale. The four modules are indicated at the top of the figure and shown in bold typeface, whereas domains within each module are labeled with standard (lightface) typeface. The nomenclature for modules follows that devised by Swain and Falke (67) for MCPs. Overlap between the HAMP domain HD2 and S-helix elements is indicated in gray. The three conserved Cys residues within the central module (62) are indicated. TM1 and TM2 denote the two transmembrane helices. Helices H1 to H4 of the periplasmic domain (24), and the transmitter domain H, N, D, G (79), and X (41) boxes, are labeled. The HPK 7 family of transmitter sequences, including NarX, have no F box and an unconventional G box (79). The scale bar at the bottom of the figure shows the number of aminoacyl residues.The HAMP domain functions as a signal conversion module in a variety of homodimeric proteins, including histidine protein kinases, adenylyl cyclases, MCPs, and certain phosphatases (12, 20, 77). This roughly 50-residue domain consists of a pair of amphiphilic α-helices, termed HD1 and HD2 (formerly AS1 and AS2) (67), joined by a connector (Fig. ​(Fig.2A).2A). Results from nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy, Cys and disulfide scanning, and mutational analysis converge on a model in which the HD1 and HD2 α-helices form a four-helix parallel coiled-coil (7, 20, 30, 42, 67, 75, 84). The mechanisms through which HAMP domains mediate signal conduction remain to be established (30, 42, 67, 84) (for commentary, see references 43, 49, and 50).Open in a separate windowFIG. 2.HAMP domain extensions. (A) Sequences from representative MCPs (E. coli Tsr and Salmonella enterica serovar Typhimurium Tar) and S-helix-containing sensors (E. coli NarX, NarQ, and BarA, and S. cerevisiae Sln1p). The HAMP domain, S-helix element, and the initial sequence of the MCP adaptation region are indicated. Flanking numbers denote positions of the terminal residues within the overall sequence. Sequential heptad repeats are indicated in alternating bold and standard (lightface) typeface. Numbering for heptad repeats in the methylation region and S-helix sequences has been described previously (4, 8). Numbers within the HD1 and HD2 helices indicate interactions within the HAMP domain (42). Residues at heptad positions a and d are enclosed within boxes, residues at the stutter position a/d are enclosed within a thickly outlined box, and residues in the S-helix ERT signature are in bold typeface. (B) NarX mutational alterations. Deletions are depicted as boxes, and missense substitutions are shown above the sequence. Many of these deletions were reported previously (10) and are presented here for comparison. The phenotypes conferred by the alterations are indicated as follows: impaired induction, black box; constitutive and elevated basal, light gray box; reversed response, dark gray box; wild-type, white box; null, striped box.Coiled-coils result from packing of two or more α-helices (27). The primary sequence of coiled-coils exhibits a characteristic heptad repeat pattern, denoted as a-b-c-d-e-f-g (52, 61), in which positions a and d are usually occupied by nonpolar residues (reviewed in references 1, 47, and 80). For example, the coiled-coil nature of the HAMP domain can be seen in the heptad repeat patterns within the HD1 and HD2 sequences (Fig. ​(Fig.2A2A).Coiled-coil elements adjacent to the HAMP domain have been identified in several sensors, including Saccharomyces cerevisiae Sln1p (69) and Escherichia coli NarX (60). Recently, this element was defined as a specific type of dimeric parallel coiled-coil, termed the signaling helix (S helix), present in a wide range of signaling proteins (8). Sequence comparisons delimit a roughly 40-residue element with a conserved heptad repeat pattern (Fig. ​(Fig.2A).2A). Based on mutational analyses of Sln1p and other proteins, the S helix is suggested to function as a switch that prevents constitutive activation of adjacent output domains (8).The term “signaling helix” previously was used to define the α4-TM2 extended helix in MCPs (23, 33). Here, we use the term S helix to denote the element described by Anantharaman et al. (8).The NarX and NarQ sensors encompass four distinct modules (Fig. ​(Fig.1):1): the amino-terminal transmembrane signaling module, the signal conversion module (including the HAMP domain and S-helix element), the central module of unknown function, and the carboxyl-terminal transmitter module (62). The S-helix element presumably functions together with the HAMP domain in conducting ligand-responsive motions from the transmembrane signaling module to the central module, ultimately regulating transmitter module activity.Regulatory output by two-component sensors reflects opposing transmitter activities (reviewed in reference 55). Positive function results from transmitter autokinase activity; the resulting phosphosensor serves as a substrate for response regulator autophosphorylation. Negative function results from transmitter phosphatase activity, which accelerates phosphoresponse regulator autodephosphorylation (reviewed in references 64 and 65). We envision a homogeneous two-state model for NarX (17), in which the equilibrium between these mutually exclusive conformations is modulated by ligand-responsive signaling.Previous work from our laboratory concerned the NarX and other HAMP domains (9, 10, 26, 77) and separately identified a conserved sequence in NarX and NarQ sensors, the Y box, that roughly corresponds to the S helix (62). Therefore, we were interested to explore the NarX S helix and to test some of the predictions made for its function. Results show that the S helix is critical for signal conduction and suggest that it functions as an extension of the HAMP HD2 α-helix in a subset of sensors exemplified by Sln1p and NarX. Moreover, a stutter discontinuity in the heptad repeat pattern was found to be essential for the NarX response to signal and to be conserved in several distinct classes of HAMP-containing sensors.     

Long-Term Fish Oil Supplementation Induces Cardiac Electrical Remodeling by Changing Channel Protein Expression in the Rabbit Model

Clinical trials and epidemiological studies have suggested that dietary fish oil (FO) supplementation can provide an anti-arrhythmic benefit in some patient populations. The underlying mechanisms are not entirely clear. We wanted to understand how FO supplementation (for 4 weeks) affected the action potential configuration/duration of ventricular myocytes, and the ionic mechanism(s)/molecular basis for these effects. The experiments were conducted on adult rabbits, a widely used animal model for cardiac electrophysiology and pathophysiology. We used gas chromatography - mass spectroscopy to confirm that FO feeding produced a marked increase in the content of n-3 polyunsaturated fatty acids in the phospholipids of rabbit hearts. Left ventricular myocytes were used in current and voltage clamp experiments to monitor action potentials and ionic currents, respectively. Action potentials of myocytes from FO-fed rabbits exhibited much more positive plateau voltages and prolonged durations. These changes could be explained by an increase in the L-type Ca current (I_CaL) and a decrease in the transient outward current (I_to) in these myocytes. FO feeding did not change the delayed rectifier or inward rectifier current. Immunoblot experiments showed that the FO-feeding induced changes in I_CaL and I_to were associated with corresponding changes in the protein levels of major pore-forming subunits of these channels: increase in Cav1.2 and decrease in Kv4.2 and Kv1.4. There was no change in other channel subunits (Cav1.1, Kv4.3, KChIP2, and ERG1). We conclude that long-term fish oil supplementation can impact on cardiac electrical activity at least partially by changing channel subunit expression in cardiac myocytes.     

The dynamics of microbial partnerships in the coral Isopora palifera

Both bacteria and algal symbionts (genus Symbiodinium), the two major microbial partners in the coral holobiont, respond to fluctuations in the environment, according to current reports; however, little evidence yet indicates that both populations have any direct interaction with each other in seasonal fluctuation. In this study, we present field observations of a compositional change in bacteria and Symbiodinium in the coral Isopora palifera in three separate coral colonies following monthly sampling from February to November in 2008. Using massively parallel pyrosequencing, over 200 000 bacterial V6 sequences were classified to build the bacterial community profile; in addition, the relative composition and quantity of Symbiodinium clades C and D were determined by real-time PCR. The results showed that coral-associated bacterial and Symbiodinium communities were highly dynamic and dissimilar among the tagged coral colonies, suggesting that the effect of host specificity was insignificant. The coral-associated bacterial community was more diverse (Shannon index up to 6.71) than previous estimates in other corals and showed rapid seasonal changes. The population ratios between clade C and D groups of Symbiodinium varied in the tagged coral colonies through the different seasons; clade D dominated in most of the samples. Although significant association between bacteria and symbiont was not detected, this study presents a more detailed picture of changes in these two major microbial associates of the coral at the same time, using the latest molecular approaches.