Passive Approximator Vascular Closure Device Use in Patients with Shallow Femoral Artery Depth Increases Puncture-site Complications in Neuroendovascular Treatment.

OBJECTIVE: Puncture-site complications in interventional radiology sometimes cause severe conditions. Vascular closure devices play an important role in preventing puncture-site complications. Vascular closure devices are divided into 2 types, the directly suturing or clipping type (active approximators) and adherent sealant types (passive approximators). However, which types of vascular closure device are the safest and most effective for achieving hemostasis remains unclear. We analyzed the efficacy of each type of vascular closure device and risk factors for puncture-site complications. METHODS: This study investigated 327 consecutive cases of neuroendovascular surgery using a transfemoral procedure during a 2-year study period. Passive approximators (Angioseal [St Jude Medical, Saint Paul, MN] and Exoseal [Cordis Corporation, Miami, FL]) were mainly used in the first half and active approximators (Perclose [Abbot Vascular, Santa Clara, CA]) in the second. We compared groups and estimated risk factors for puncture-site complications. RESULTS: All procedures were successful. Comparing groups with and without puncture-site complications, use of passive approximators and ≥3 antithrombotic medications tended to be more frequent and distance from skin to femoral artery and body mass index tended to be lower in the group with complications without significance. The cutoff for femoral artery depth calculated from a receiver operating characteristic curve was 16.43 mm. Multivariate analysis revealed ≥3 antithrombotic medications (P = 0.002, OR 15.29, 95% CI 2.76-85.76) and passive approximator use in patients with femoral artery depth <16.43 mm (P < 0.001, OR 17.08, 95% CI 2.95-57.80) were significantly higher in the group with puncture-site complications. CONCLUSIONS: Passive approximator use in patients with shallow femoral artery depth increases puncture-site complications in neuroendovascular treatment.

Malignant Transformation and Leptomeningeal Melanomatosis in a Primary Meningeal Melanocytoma: A Case Report and Review of Literature.

Meningeal melanocytomas of the central nervous system, although typically benign, rarely undergo malignant transformations. A 46-year-old man presented with headache and nausea 4 years after gross total resection of a craniovertebral junction meningeal melanocytoma at another hospital. The initial clinical course was previously reported.1) Computed tomography revealed the presence of multiple intracranial mass lesions. Furthermore, magnetic resonance imaging showed multiple intracranial lesions and meningeal dissemination. A biopsy was performed for a circumflex lesion located in the right frontal lobe. Pathological examination showed anaplastic changes and a Ki-67 index of 33%. Based on the pleomorphic changes and high mitotic activity, the patient was diagnosed with primary cerebral malignant melanoma. The patient received four cycles of nivolumab (80 mg) and ipilimumab (165 mg), followed by whole-brain radiotherapy (37.5 Gy). However, the disease progressed after the third cycle. Genome analysis revealed GNAQ Q209P and SF3B1 R625C mutations, but no treatments related to these gene mutations were available. Despite the seven cycles of nivolumab therapy, the patient eventually passed away 9 months after surgery. This case was a rare example of malignant transformation and leptomeningeal melanomatosis in a meningeal melanocytoma. It highlights the importance of careful follow up after gross total resection. Identification of molecular alterations can lead to better detection of melanocytic melanomas with poor prognosis and high risk of recurrence and metastasis. It can also facilitate the development of novel therapeutic options for these patients.

Fatal case of TAFRO syndrome with unilateral adrenal hemorrhage in early-stage disease.

Thrombocytopenia, anasarca, fever, reticulin fibrosis/renal failure, and organomegaly comprise TAFRO syndrome, which was proposed as a distinct clinical entity from iMCD without TAFRO syndrome (iMCD-NOS) due to its aggressive clinical course, refractoriness to corticosteroids, presence of thrombocytopenia, increased level of alkaline phosphatase, and normal level of gammaglobulin. However, diagnosing TAFRO syndrome in its early stages is challenging because it is rare and its diagnostic criteria are complicated. We describe a patient with TAFRO syndrome and adrenal hemorrhage who demonstrated a rapid decline in her clinical condition and did not respond to steroid pulse therapy, resulting in a fatal outcome. In the early stage of her clinical course, she developed unilateral adrenal hemorrhage with mild thrombocytopenia and normal clotting times, suggesting adrenal hemorrhage as a unique manifestation of TAFRO syndrome. In general, patients with TAFRO syndrome exhibit a more aggressive clinical course and poorer outcome than those with iMCD-NOS. To ameliorate this poor prognosis, it is important to diagnose the disease early and immediately start powerful immunosuppressive agents such as tocilizumab. Based on this case, adrenal hemorrhage may suggest TAFRO syndrome, and facilitate the rapid diagnosis of this complicated and rare disease.

Physicochemical Properties of the Mammalian Molecular Chaperone HSP60.

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.

The regulation mechanisms of AhR by molecular chaperone complex.

The AhR, so called the dioxin receptor, is a member of the nuclear receptor superfamily. The ligand-free AhR forms a cytosolic protein complex with the molecular chaperone HSP90, co-chaperone p23, and XAP2 in the cytoplasm. Following ligand binding like 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the AhR translocates into the nucleus. Although it has been reported that HSP90 regulates the translocation of the AhR to the nucleus, the precise activation mechanisms of the AhR have not yet been fully understood. AhR consists of the N-terminal bHLH domain containing NLS and NES, the middle PAS domain and the C-terminal transactivation domain. The PAS domain is familiar as a ligand and HSP90 binding domain. In this study, we focused on the bHLH domain that was thought to be a HSP90 binding domain. We investigated the binding properties of bHLH to HSP90. We analyzed the direct interaction of bHLH with HSP90, p23 and XAP2 using purified proteins. We found that not only the PAS domain but also the bHLH domain bound to HSP90. The bHLH domain forms complex with HSP90, p23 and XAP2. We also determined the bHLH binding domain was HSP90 N-domain. The bHLH domain makes a complex with HSP90, p23 and XAP2 via the HSP90 N-domain. Although the NLS is closed in the absence of a ligand, the structure of AhR will be changed in the presence of a ligand, which leads to NLS open, result in the nuclear translocation of AhR.

HSP60 possesses a GTPase activity and mediates protein folding with HSP10.

The mammalian molecular chaperone, HSP60, plays an essential role in protein homeostasis through mediating protein folding and assembly. The structure and ATP-dependent function of HSP60 has been well established in recent studies. After ATP, GTP is the major cellular nucleotide. In this paper, we have investigated the role of GTP in the activity of HSP60. It was found that HSP60 has different properties with respect to allostery, complex formation and protein folding activity depending on the nucleoside triphosphate present. The presence of GTP slightly affected the ATPase activity of HSP60 during protein folding. These results provide clues as to the functional mechanism of the HSP60-HSP10 complex.

Use of a digital phantom developed by QIBA for harmonizing SUVs obtained from the state-of-the-art SPECT/CT systems: a multicenter study.

BACKGROUND: Although quantitative analysis using standardized uptake value (SUV) becomes realistic in clinical single-photon emission computed tomography/computed tomography (SPECT/CT) imaging, reconstruction parameter settings can deliver different quantitative results among different SPECT/CT systems. This study aims to propose a use of the digital reference object (DRO), which is a National Electrical Manufacturers Association (NEMA) phantom-like object developed by the Quantitative Imaging Biomarker Alliance (QIBA) fluorodeoxyglucose-positron emission tomography technical committee, for the purpose of harmonizing SUVs in Tc-99m SPECT/CT imaging. METHODS: The NEMA body phantom with determined Tc-99m concentration was scanned with the four state-of-the-art SPECT/CT systems. SPECT data were reconstructed using different numbers of the product of subset and iteration numbers (SI) and the width of 3D Gaussian filter (3DGF). The mean (SUVmean), maximal (SUVmax), and peak (SUVpeak) SUVs for six hot spheres (10, 13, 17, 22, 28, and 37 mm) were measured after converting SPECT count into SUV using Becquerel calibration factor. DRO smoothed by 3DGF with a FWHM of 17 mm (DRO17 mm) was generated, and the corresponding SUVs were measured. The reconstruction condition to yield the lowest root mean square error (RMSE) of SUVmeans for all the spheres between DRO17 mm and actual phantom images was determined as the harmonized condition for each SPECT/CT scanner. Then, inter-scanner variability in all quantitative metrics was measured before (i.e., according to the manufacturers' recommendation or the policies of their own departments) and after harmonization. RESULTS: RMSE was lowest in the following reconstruction conditions: SI of 100 and 3DGF of 13 mm for Brightview XCT, SI of 160 and 3DGF of 3 pixels for Discovery NM/CT, SI of 60 and 3DGF of 2 pixels for Infinia, and SI of 140 and 3DGF of 15 mm for Symbia. In pre-harmonized conditions, coefficient of variations (COVs) among the SPECT/CT systems were greater than 10% for all quantitative metrics in three of the spheres, SUVmax and SUVmean, in one of the spheres. In contrast, all metrics except SUVmax in the 17-mm sphere yielded less than 10% of COVs after harmonization. CONCLUSIONS: Our proposed method clearly reduced inter-scanner variability in SUVs. A digital phantom developed by QIBA would be useful for harmonizing SUVs in multicenter trials using SPECT/CT.

High dose of antibiotic colistin induces oligomerization of molecular chaperone HSP90.

Colistin is an antimicrobial cationic peptide that belongs to the polymyxin family. Colistin was clinically used for the treatment of gram-negative infections but fell out of favour because of its significant side effects including neurotoxicity and nephrotoxicity. More recently, colistin has been regarded as one of the important options for nosocomial infections caused by multidrug resistant bacteria. Mechanisms of both the side effect onset of the drug and the side effect reduction are yet to be elucidated. In this study, we identified the specific binding protein of colistin using an affinity column chromatography. Colistin binds to the molecular chaperone HSP90. Although colistin slightly suppressed the chaperone activity of HSP90, there are no effects on the ATPase activity for a low concentration of colistin. Interestingly, colistin-induced aggregation of HSP90 via the N-domain. As for the cell viability of the SHSY5Y cell, the cell viability decreased to approximately 80% by the colistin 300 µM. However, the cell viability recovered to approximately 100% by adding ATP dosage. The same result was obtained by dot blot assay using anti-HSP90 antibody. Our results may help to understand the side effect mechanism of colistin.

NIP-SNAP-1 and -2 mitochondrial proteins are maintained by heat shock protein 60.

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2.

Functional structure and physiological functions of mammalian wild-type HSP60.

The Chaperonins comprise a family of molecular chaperones having a double-ring structure and similar sequence homology. These proteins play an essential role in biological reactions that mediate the folding of newly synthesized polypeptides and partially denatured proteins. In the prokaryotic group I chaperonins, structural and reaction cycle analyses of GroEL and its co-chaperone GroES have been performed in detail. While in eukaryotes, there have been limited reports analyzing the group I chaperonin HSP60 and its co-chaperone HSP10. In the present study, we purified the wild type HSP60 from porcine liver and investigated the interaction between HSP60 and HSP10, including conformation and physiological relationships. Based on the results of transmission electron microscopy, native PAGE, and gel filtration column chromatography, the wild type HSP60 displayed a heptameric single-ring structure in the absence of ATP. In contrast, HSP60 formed mainly a "football-type" complex with HSP10 in the presence of ATP and mediated the refolding of denatured substrate protein. The functional conformation cycle of the purified mammalian HSP60 is distinct from the cycle of the prokaryotic GroEL/GroES chaperonin.

Geranylgeranylacetone selectively binds to the HSP70 of Helicobacter pylori and alters its coccoid morphology.

Geranylgeranylacetone (GGA) is used to treat patients suffering from peptic ulcers and gastritis. We examined the effect of GGA on Helicobacter pylori, which is a causative factor of gastrointestinal diseases. Previously, we have reported that GGA binds specifically to the molecular chaperone HSP70. In this paper, we report that GGA bounds to H. pylori HSP70 (product of the DnaK gene) with 26-times higher affinity than to human HSP70, and induced large conformational changes as observed from surface plasmon resonance and circular dichroism. Binding of GGA suppressed the activity of the H. pylori chaperone. GGA also altered several characteristics of H. pylori cells. GGA-treated cells elicited enhanced interleukin-8 production by gastric cancer cell lines and potentiated susceptibility to complement as compared to untreated cells. GGA also caused morphological alterations in H. pylori as reflected in fewer coccoid-like cells, suggesting that GGA converts H. pylori to an actively dividing, spiral state (vegetative form) from a non-growing, coccoid state. This morphological conversion by GGA resulted in accelerated growth of H. pylori. These results suggest a model in which GGA sensitizes H. pylori to antibiotic treatment by converting the cells to an actively growing state.

The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90.

The aryl hydrocarbon receptor is a member of the nuclear receptor superfamily that associates with the molecular chaperone HSP90 in the cytoplasm. The activation mechanism of the AhR is not yet fully understood. It has been proposed that after binding of ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3methylcholanthrene (3-MC), or ß-naphthoflavone (ß-NF), the AhR dissociates from HSP90 and translocates to the nucleus. It has also been hypothesized that the AhR translocates to the nucleus and forms a complex with HSP90 and other co-chaperones. There are a few reports about the direct association or dissociation of AhR and HSP90 due to difficulties in purifying AhR. We constructed and purified the PAS domain from AhR. Binding of the AhR-PAS domain to ß-NF affinity resin suggested that it possesses ligand-binding affinity. We demonstrated that the AhR-PAS domain binds to HSP90 and the association is not affected by ligand binding. The ligand 17-DMAG inhibited binding of HSP90 to GST-PAS. In an immunoprecipitation assay, HSP90 was co-immunoprecipitated with AhR both in the presence or absence of ligand. Endogenous AhR decreased in the cytoplasm and increased in the nucleus of HeLa cells 15 min after treatment with ligand. These results suggested that the ligand-bound AhR is translocated to nucleus while in complex with HSP90. We used an in situ proximity ligation assay to confirm whether AhR was translocated to the nucleus alone or together with HSP90. HSP90 was co-localized with AhR after the nuclear translocation. It has been suggested that the ligand-bound AhR was translocated to the nucleus with HSP90. Activated AhR acts as a transcription factor, as shown by the transcription induction of the gene CYP1A1 8 h after treatment with ß-NF.

Zinc-L-carnosine binds to molecular chaperone HSP70 and inhibits the chaperone activity of the protein.

In this study, we have investigated the specific binding proteins of Zinc-L-carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro. A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0-1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70.