C-terminal amino acids in the type I transmembrane domain of L-type lectin VIP36 affect γ-secretase susceptibility.

Regulated intramembrane proteolysis (RIP) is a two-step processing mechanism for transmembrane proteins consisting of ectodomain shedding (shedding), which removes the extracellular domain through juxtamembrane processing and intramembrane proteolysis, which processes membrane-anchored shedding products within the transmembrane domain. RIP irreversibly converts one transmembrane protein into multiple soluble proteins that perform various physiological functions. The only requirement for the substrate of γ-secretase, the major enzyme responsible for intramembrane proteolysis of type I transmembrane proteins, is the absence of a large extracellular domain, and it is thought that γ-secretase can process any type I membrane protein as long as it is shed. In the present study, we showed that the shedding susceptible type I membrane protein VIP36 (36 kDa vesicular integral membrane protein) and its homolog, VIPL, have different γ-secretase susceptibilities in their transmembrane domains. Analysis of the substitution mutants suggested that γ-secretase susceptibility is regulated by C-terminal amino acids in the transmembrane domain. We also compared the transmembrane domains of several shedding susceptible membrane proteins and found that each had a different γ-secretase susceptibility. These results suggest that the transmembrane domain is not simply a stretch of hydrophobic amino acids but is an important element that regulates membrane protein function by controlling the lifetime of the membrane-anchored shedding product.

Severe hyponatremia with seizures and confirmed mild brain edema by hysteroscopic myomectomy: a case report.

BACKGROUND: Hyponatremia can be developed during hysteroscopic surgery with electrolyte-free irrigation fluid. We experienced severe hyponatremia with postoperative seizures and confirmed mild brain edema. CASE PRESENTATION: A quadragenarian female patient underwent a 2-h hysteroscopic myomectomy with electrolyte-free fluid for uterine distension under general anesthesia. Plasma sodium level of 84.1 mmol/L 100 min after the start of surgery indicated excessive absorption of the irrigation fluid. Acute severe hyponatremia was diagnosed with significant edema in the conjunctiva, lip, and extremities. She was treated with a continuous infusion of hypertonic saline. However, seizures and cerebral edema developed 7 h later. The patient recovered without neurological deficits at postoperative day 2. CONCLUSION: The electrolyte-free irrigation fluid can be absorbed rapidly during hysteroscopic surgery. Its interruption with hyponatremia should be considered against prolonged surgery. Especially under general anesthesia, caution should be exercised because the typical symptoms of hyponatremia such as nausea and confusion are blinded.

The constitutive high-affinity Met-binding site in the kringle domain is dispensable for the signalling activity of hepatocyte growth factor.

Activation of a tyrosine kinase receptor Met by hepatocyte growth factor (HGF) requires binding of proteolytically activated, two-chain (tc) HGF, but the biochemical detail of this ligand-receptor interaction specificity remains elusive because biologically inactive single chain (sc) HGF can also bind to Met with high affinity. We found that this proteolysis-independent Met binding can be eliminated by mutagenesis introduced in the kringle domain without losing the ability to bind and activate cellular Met receptor after proteolytic activation, arguing against this site's involvement in the physiological signalling. This non-signal producing Met-HGF interaction can also be eliminated by addition of a heparin mimetic sucrose octasulphate (SOS). By including SOS in the running buffer, we succeeded in detecting cleavage-dependent tcHGF-Met complex formation by size exclusion chromatography.

An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system.

Peptide-based affinity tags are commonly used in recombinant production/purification of proteins, and are often preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal antibody 2H5 recognizing an undecapeptide tag called "eTev", which contains a recognition sequence for Tobacco Etch Virus (TEV) protease. In the crystal structure of 2H5-eTev complex, the long eTev peptide assumes compact α-helical conformation in the binding groove, exposing both ends to the solution. This architecture allowed us to connect eTev with another peptide tag called PA tag via linker sequence, ensuring the simultaneous access of two anti-tag antibodies. When this tandem double tag was attached at one end of various proteins, it enabled highly sensitive and protein-independent detection by sandwich ELISA. Utilizing this system during a rapid cell line screening, we succeeded in isolating stable cell clones expressing high level of mouse Wise protein.

Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability.

Antibody fragments are frequently used as a "crystallization chaperone" to aid structural analysis of complex macromolecules that are otherwise crystallization resistant, but conventional fragment formats have not been designed for this particular application. By fusing an anti-parallel coiled-coil structure derived from the SARAH domain of human Mst1 kinase to the variable region of an antibody, we succeeded in creating a novel chimeric antibody fragment of ∼37 kDa, termed "Fv-clasp," which exhibits excellent crystallization compatibility while maintaining the binding ability of the original IgG molecule. The "clasp" and the engineered disulfide bond at the bottom of the Fv suppressed the internal mobility of the fragment and shielded hydrophobic residues, likely contributing to the high heat stability and the crystallizability of the Fv-clasp. Finally, Fv-clasp antibodies showed superior "chaperoning" activity over conventional Fab fragments, and facilitated the structure determination of an ectodomain fragment of integrin α6ß1.

Conformational Freedom of the LRP6 Ectodomain Is Regulated by N-glycosylation and the Binding of the Wnt Antagonist Dkk1.

LDL-receptor-related protein 6 (LRP6) is a single-pass membrane glycoprotein with a large modular ectodomain and forms a higher order signaling platform upon binding Wnt ligands on the cell surface. Although multiple crystal structures are available for fragments of the LRP6 ectodomain, we lack a consensus view on the overall molecular architecture of the full-length LRP6 and its dynamic aspects. Here, we used negative-stain electron microscopy to probe conformational states of the entire ectodomain of LRP6 in solution and found that the four-module ectodomain undergoes a large bending motion hinged at the junction between the second and the third modules. Importantly, the extent of inter-domain motion is modulated by evolutionarily conserved N-glycan chains proximal to the joint. We also found that the LRP6 ectodomain becomes highly compact upon complexation with the Wnt antagonist Dkk1, suggesting a potential role for the ectodomain conformational change in the regulation of receptor oligomerization and signaling.

Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies.

HGF-Met signaling contributes to various biological events by controlling cell migration. Since the abnormal activation of Met receptor causes cancer progression, inhibitors such as neutralizing antibodies are regarded as promising therapeutics. HGF is secreted as a single-chain (sc) precursor and is processed by extracellular proteases to generate disulfide-bonded two-chain (tc) HGF. Although this proteolytic processing of HGF is necessary for its biological activity, exactly how the proteolysis leads to the conversion of HGF to the active form is still unclar due to the lack of structural information. In order to gain insights about this point, we generated 6 antibodies against HGF. All antibodies recognized different epitopes on the native HGF protein and showed distinct effects when tested in a cell-based HGF-Met signaling assay. They included one antibody (t1E4) that strongly blocks Met activation by tcHGF, as well as one antibody (t8E4) exclusively recognizing the active tcHGF but not inactive scHGF. Thus, a panel of anti-HGF antibodies suitable for probing the structural mechanism of HGF activation were obtained.

Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin.

Wnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestical stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, afamin complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years.

Characterization of enterohemorrhagic Escherichia coli O111 and O157 strains isolated from outbreak patients in Japan.

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx2 and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx2 isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx1, stx2, and stx1 stx2), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx2 to an stx-negative strain may have occurred during infection.

A structure-based model of substrate discrimination by a noncanonical PDZ tandem in the intramembrane-cleaving protease RseP.

During the extracytoplasmic stress response in Escherichia coli, the intramembrane protease RseP cleaves the anti-σ(E) protein RseA only after the membrane-anchored protease DegS truncates the periplasmic part of RseA that suppresses the action of RseP. Here we analyzed the three-dimensional structure of the two tandemly arranged PSD-95/Dlg/ZO-1 (PDZ) domains (PDZ tandem) present in the periplasmic region of RseP and revealed that the two putative ligand-binding grooves constitute a single pocket-like structure that would lie just above the active center sequestrated within the membrane. Complete removal of the PDZ tandem from RseP led to the intramembrane cleavage of RseA without prior truncation by DegS. Furthermore, mutations expected to destabilize the tertiary structure of the PDZ tandem also caused the deregulation of the sequential cleavage. These observations suggest that the PDZ tandem serves as a size-exclusion filter to accommodate the truncated form of RseA into the active center.

[Causes of emergency hospitalization of elderly diabetic patients].

In recent years there has been a rise in the number of diabetic patients in Japan, with the increase in elderly diabetic patients becoming a serious problem. This study looked at 488 elderly type 2 diabetes patients who were admitted as emergency cases to the Department of Internal Medicine, JA Yoshida General Hospital, Akitakada City, Japan. All patients were classified by age into three groups: <70, 70-80 and >80. The most common cause of emergency hospitalization in each of the three age groups was infection. This was significantly higher in the >80 group in comparison with<70 (p<0.05). The most common infection among the three groups was respiratory infection, followed by urinary tract infection. The number of emergency hospitalization cases due to hypoglycemia was much higher in the over 80 group, particularly in comparison with<70-80 (p<0.05). The incidence of hypoglycemia in our patients could be explained mainly by reduced energy intake. Most cases were treated with oral administration of hypoglycemic drugs. As elderly diabetic patients have a number of underlying illnesses that are prone to aggravation and may lead to unfavorable prognosis, early medical examination and disease detection are considered to be important. Pharmacists are required to educate patients, home-visit nursing care personnel on sick-day rule, and provide diabetes care.

Phenotypic and genetic analyses of Campylobacter jejuni Lior serotype 76 isolated from chicken meat and clinical specimens.

The aim of this study was to examine the link between Campylobacter jejuni isolates obtained from chicken meat (n = 7) and gastroenteritis patients (n = 744). In total, 751 isolates were subjected to Lior serotyping. All the isolates from chicken meats were serotyped as Lior serotype 76 (LIO76). Among 23 of the identified LIO76 strains, 13 strains (6 from chicken meat and 7 from clinical specimens) were indistinguishable by Penner serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis. These strains were isolated in 2 different Japanese prefectures in 2004-2005, suggesting that chicken meat is an etiological agent of Campylobacter gastroenteritis and that a diffuse outbreak occurred during this time. Therefore, a continuous surveillance program should be established in Japan in order to prevent Campylobacter gastroenteritis, especially large-scale food-borne outbreaks.

A rapid screening method for cell lines producing singly-tagged recombinant proteins using the "TARGET tag" system.

Recombinant production of extracellular glycoproteins in stable mammalian cell lines is an ideal technique for obtaining a large quantity of high-quality proteins. In most cases, however, current methodologies do not allow for sufficiently rapid cell line development and protein purification. Here, we describe a 21-residue peptide tag (designated as TARGET tag) and its use for rapid stable cell line development and purification. The ability of the anti-tag antibody P20.1 to withstand repetitive regeneration cycles has enabled the development of a sensitive surface plasmon resonance-based screening format that requires only 20 microl of cell culture supernatants. Direct and semi-quantitative screening at the 96-well culture scale eliminated the need for a second screening, re-cloning, or sorting, thereby minimizing culture pre-production time. Using this system, "high producer" cell lines were established in less than a month, and milligram quantities of target proteins could be purified with a standardized protocol.

Detection of endogenous LRP6 expressed on human cells by monoclonal antibodies specific for the native conformation.

LRP6 is a cell surface molecule that plays a critical role in the Wnt signaling pathway, and is implicated in numerous human diseases. Studies of cellular signaling mediated by LRP6 have relied on overexpression experiments, due to the lack of good monoclonal antibodies (mAbs) reactive with native LRP6 ectodomain. By using native recombinant LRP6 ectodomain fragment produced in mammalian expression system, we succeeded in developing a panel of anti-human LRP6 mAbs. Selected mAbs were capable of staining endogenous LRP6 on cell surface by using flow cytometry and immunofluorescence microscopy, and enriching detergent-solubilized LRP6 from cell lysate by immunoprecipitation.

Novel affinity tag system using structurally defined antibody-tag interaction: application to single-step protein purification.

Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody-peptide interaction, opening opportunities for further improvements/modifications.

[Relation between comprehensive-geriatric assessment and the ability of self-administration in elderly diabetic patients].

Intellectual ability of self-administration plays a crucial role in a diabetes regimen. However, in many cases, self-administration is considered difficult, because of the impairment of activities of the individual's daily living (ADL), instrumental ADL and cognitive function. To assess comprehensive-geriatric-assessment (CGA) in elderly diabetic sufferers, 62 elderly inpatients and outpatients aged over 70 years were investigated. CGA includes Barthel index (BI), Mini-mental-state-examination (MMSE) and the Tokyo Metropolitan Institute of Gerontology index of competence (TMIGIC). The relation of ability to self-administer and CGA was examined. In oral therapy, there was significant difference between self-administration and non-self-administration in MMSE (p=0.0065), BI (p=0.0219) and TMIGIC (p=0.0053). Among these indexes, TMIGIC was the most sensitive index in oral therapy. In insulin therapy, there was also significant difference between self-administration and non-self-administration: MMSE (p=0.00042), BI (p=0.000019) and TMIGIC (p=0.0019). Among these indexes, BI was the most sensitive index in insulin therapy. It was suggested that CGA was useful to assess the ability of self-administration in elderly diabetic patients.

Malaria prevention and stand-by emergency treatment among Japanese travelers.

BACKGROUND: While chemoprophylaxis remains the safest option for most travelers to malaria high-risk areas, stand-by emergency treatment (SBET) may also be a sensible option especially for travelers to low-risk areas, due to the possible adverse effects (AEs) of prophylactic antimalarials. However, studies on the suitability of SBET actually implemented by travelers are scarce, especially those targeting Japanese travelers. We investigated to what extent malaria prevention measures are taken and how effectively SBET is used by Japanese travelers to malaria-endemic areas. MATERIALS AND METHODS: A questionnaire study was conducted targeting Japanese travelers who visited quarantine stations for pre-travel health advice and who had previously visited malaria-endemic areas as defined by the World Health Organization (N = 160). RESULTS: The results showed that only a minority (13%) of travelers to malaria-endemic areas took chemoprophylaxis. Although most (89%) of the SBET users (N = 9) took antimalarial drugs when they experienced both fever and chills, characteristic of clinical malaria, there were several problems related to SBET. Some (22%) of the subjects conducted SBET less than 7 days after entering the area, most (89%) of them did so when a medical facility was readily accessible, and many (56%) failed to seek medical attention soon after SBET or did not at all. CONCLUSIONS: Japanese travelers to malaria-endemic areas seemed less protected with the use of chemoprophylaxis. Furthermore, problems related to SBET among Japanese travelers were identified. These should be taken into full consideration when educating both travelers and travel health professionals to avoid risks of malaria and possible AEs of antimalarial drugs.