Structure and dynamics of polymyxin-resistance-associated response regulator PmrA in complex with promoter DNA.

PmrA, an OmpR/PhoB family response regulator, manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB response regulator induces the formation of a symmetric dimer in the N-terminal receiver domain (REC), promoting two C-terminal DNA-binding domains (DBDs) to recognize promoter DNA to elicit adaptive responses. Recently, determination of the KdpE-DNA complex structure revealed an REC-DBD interface in the upstream protomer that may be necessary for transcription activation. Here, we report the 3.2-Å-resolution crystal structure of the PmrA-DNA complex, which reveals a similar yet different REC-DBD interface. However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC-DBD interaction is transiently populated in solution. Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression. We propose that REC-DBD interdomain dynamics and the DBD-DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

Backbone resonance assignments of the 54 kDa dimeric C-terminal domain of murine STING in complex with DMXAA.

The mammalian ER protein STING (stimulators of interferon genes) is an important innate immunity protein for linking detection of novel secondary messengers c-di-GMP, c-di-AMP, cGAMP or cytosolic dsDNA to the activation of TANK kinase 1 and its downstream interferon regulator factor 3. Recently quite a few of crystal structures representing different states of the C-terminal domain (CTD) of human and murine STING (hSTING and mSTING) in complex with c-di-GMP, cGAMP or DMXAA have been reported. However, the C-terminal 42 residues of STING-CTD, which may be important in mediating the downstream reactions, is invisible or absent in all reported X-ray structures. In addition, X-ray crystal structures may be subject to crystal packing force. Hence an alternate method of determining the structure and function of STING in a near physiological condition is essential. We now report the near complete backbone resonance assignments of the 54 kDa dimeric mSTING-CTD in complex with DMXAA, which is the first step in determining its complex structure and understanding why DMXAA, which is a very efficient agent for curing mouse cancer, is totally ineffective in human.

Solution structure and tandem DNA recognition of the C-terminal effector domain of PmrA from Klebsiella pneumoniae.

Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC) recognizes tandem imperfect repeat sequences on the promoters of genes to induce antimicrobial peptide resistance after phosphorylation and dimerization of its N-terminal receiver domain (PmrAN). However, structural information concerning how phosphorylation of the response regulator enhances DNA recognition remains elusive. To gain insights, we determined the nuclear magnetic resonance solution structure of PmrAC and characterized the interactions between PmrAC or BeF3(-)-activated full-length PmrA (PmrAF) and two DNA sequences from the pbgP promoter of K. pneumoniae. We showed that PmrAC binds to the PmrA box, which was verified to contain two half-sites, 5'-CTTAAT-3' and 5'-CCTAAG-3', in a head-to-tail fashion with much stronger affinity to the first than the second site without cooperativity. The structural basis for the PmrAC-DNA complex was investigated using HADDOCK docking and confirmed by paramagnetic relaxation enhancement. Unlike PmrAC, PmrAF recognizes the two sites simultaneously and specifically. In the PmrAF-DNA complex, PmrAN may maintain an activated homodimeric conformation analogous to that in the free form and the interactions between two PmrAC molecules aid in bending and binding of the DNA duplex for transcription activation.

Solution structure of the C-terminal NP-repeat domain of Tic40, a co-chaperone during protein import into chloroplasts.

Chloroplasts protein precursors translated in the cytosol traverse the membranes to reach their intended destination with the help of translocon complexes called translocon at the outer envelope of chloroplasts and translocon at the inner envelope of chloroplasts (TIC), respectively. Two components of the TIC translocon, Tic110 and Tic40, which combine with Hsp93 (ClpC), are involved in protein translocation across the inner membrane into the stroma. The C-terminal NP-repeat domain of Tic40 (Tic40-NP) is homologous to the DP-repeat domain of co-chaperones Hsp70-interacting and Hsp70/Hsp90-organizing proteins. Interaction of Tic40-NP and Hsp93 stimulates ATP hydrolysis of Hsp93, but the hydrolysis is abolished in both N320A and N329A mutants of Tic40-NP. Here, we determined the nuclear magnetic resonance structure of Tic40-NP, which mainly consists of five α-helices stabilized by two hydrophobic cores. In addition, chemical shift perturbation results suggested that some residues at α1 and α5, as well as residues Asn320 and Asn329, cause conformational change on the two mutants, which may subsequently affect their binding to Hsp93. We provide valuable information for further investigating how Tic40-NP interacts with Hsp93.

Association between cerebral microbleeds and prior primary intracerebral hemorrhage in ischemic stroke patients.

OBJECTIVE: To investigate association between cerebral microbleeds (CMB) and prior intracerebral hemorrhage (ICH) on MRI and topographic correlation of the two types of lesions. PATIENTS AND METHODS: Two hundred and sixty consecutive patients (67.0+/-11.1 years) with ischemic stroke were included. CMB and prior ICH were assessed on T2-gradient-echo MRI. The presence and number of CMB as predictors for prior ICH were examined. Topographic correlations between CMB and ICH lesions in patients with prior ICH in the infratentorial, basal ganglionic/thalamic and cortico-subcortical regions were tested. RESULTS: CMB were observed in 113 (43.5%) patients and a total of 50 prior primary ICH lesions were observed in 39 (15.0%) patients. Among the ICH lesions, 39 (78%) were asymptomatic. Presence of CMB (odds ratio 2.53, p=0.015) and number of CMB (odds ratio 1.11, p<0.001) were independent determinants for prior ICH. Topographic correlation between CMB and ICH was significant in the basal ganglionic/thalamic region (p=0.017), but not in the infratentorial (p=0.548) or cortico-subcortical regions (p=0.389). CONCLUSION: CMB were associated with prior ICH on MRI of patients with ischemic stroke. CMB in the basal ganglion or thalamus was associated with prior ICH in the same region.

Type II cryoglobulinemia and brain hemorrhage.

By virtue of an understanding of hemostasis and coagulopathy using modern techniques, the exact role of individual serum protein in vascular thrombosis or hemorrhage becomes more apparent. Cryoglobulin causes vasculitude and thrombosis in various vascular beds, but its role in brain hemorrhage is unknown. We encountered a cryoglobulinemic patient to have cryoglobulinemia, hypocomplementia, and cerebellar hemorrhage during a reactivation of cytomegalovirus infection. Because cryoglobulin is harmful to vessel and hemostasis, and often increases nonspecifically in response to incitement, its weight in vascular syndrome must seriously be reviewed. Coagulopathy in a reactivation of latent virus such as cytomegalovirus should be cautioned in older patients.

Hyperhomocysteinemia relates to the subtype of antiphospholipid antibodies in non-SLE patients.

Abnormal increases of antiphospholipid antibody and plasma homocysteine levels are recently emerging as nonlipidic risk factors for cerebral atherogenesis and thrombosis. Both antiphospholipid antibody and homocysteine share many similar bioeffects in hemostasis, but their interaction is still inconsistent. In this study, we examined the relation between the plasma homocysteine level and lupus anticoagulant, anticardiolipin antibody, and anti-beta2-glycoprotein I antibody in patients with noncardiac cerebral ischemia. Systemic lupus erythrematosus patients were excluded. The results showed a higher frequency of moderate hyperhomocysteinemia in patients with an abnormal increase of lupus anticoagulant only. Neither the serum folate and cobalamin levels nor methylenetetrahydrofolate reductase allele mutation contributes to this result. Accordingly, homocysteine interacts with lupus anticoagulant to promote cerebral atherosclerosis and ischemia. The role of vasculopathic or prothrombotic autoantibody generation in response to specific pathological change such as hyperhomocysteinemia warrants further investigation.

Delayed catastrophic antiphospholipid syndrome after massive cerebral infarction.

INTRODUCTION: Catastrophic antiphospholipid syndrome (CAPS) is a life-threatening disorder urgent for aggressive treatment, and its characterized systemic thromboses and coagulopathy prompt for a rapid diagnosis. CASE REPORT: A woman fell into unconsciousness two weeks after a stable course of massive cerebral ischemia was identified due to CAPS with Sjogren syndrome. Her thrombocyte count and coagulation times were was normal at initial. A high blood anti-beta(2)-glycoprotein I antibody level was found. Plasmapheresis rapidly reversed her consciousness and coagulation function. CONCLUSIONS: A normal hemostatic parameters should not exclude CAPS. A favorable prognosis depends on the rapidity of appropriate treatment.

Time course of platelet activation and von Willebrand factor in patients with non-valvular atrial fibrillation after ischemic stroke.

BACKGROUND: The present study investigated serial changes in platelet activation (expressed by CD62p) and von Willebrand factor (VWF), and the correlation between increased CD62p expression, VWF and brain infarct volume (BIV: measured by magnetic resonance imaging), and prognostic determinants in non-valvular atrial fibrillation (NVAF) patients after acute ischemic stroke (IS). METHODS AND RESULTS: CD62p expression and plasma VWF concentrations were serially measured (<48 h, on days 7, 21 and 90) using flow cytometry and enzyme-linked immunosorbent assay, respectively after acute IS in 61 NVAF patients. CD62p expression and VWF concentrations were also examined in 50 NVAF-risk control and 30 healthy individuals. The VWF concentration had no significant changes at 4 intervals among the patients and did not differ among 3 groups at acute stroke phase. CD62p expression was significantly higher in the acute phase after IS than in both control groups (both p<0.0001). However, CD62p expression declined to a significantly lower level on day 7 and to a substantially lower level thereafter (p<0.0001). CD62p expression did not differ on day 90 in the 3 groups (both p>0.5). Linear regression analysis showed that BIV and modified Rankin scale score (>3) were independently associated with increased CD62p expression (<48 h) (both p<0.01). Furthermore, the Cox proportional hazards model showed that BIV was the only independent predictor of intermediate-term (8.8+/-4.4 months) combined recurrent stroke and death. CONCLUSIONS: The CD62p expression, which reflected increased BIV, was significantly increased in NVAF patients in acute-phase IS and substantially declined thereafter. The BIV was predictive of unfavorable intermediate-term clinical outcomes.

Cholesterol granuloma of the maxillary sinus presenting as sinonasal polyp.

Cholesterol granuloma is usually found in chronic middle ear diseases. However, it rarely occurs in the sinonasal regions and only a few case reports can be found in the literature. The etiology of sinonasal cholesterol granuloma is not yet known, and the clinical manifestations are nonspecific. Most patients presented with nasal discharge. Our patient is the only reported case presenting with nasal obstruction and facial pain. Here we present a patient with cholesterol granuloma of the maxillary sinus with a nasal polyp, whose clinical, imaging, and histological characteristics were unique. The tumor was excised via the transnasal endoscopic sinus approach without recurrence after 3 years of follow-up.

An increase of blood squamous cell carcinoma antigen in Pseudomonas aeruginosa spondylitis.

Squamous cell carcinoma antigen (SCCA) is traditionally engaged for detecting and following up malignancy from a squamous cell origin. We encountered an unusual increase of blood SCCA but no other cancer markers in a patient associated with an infective lumbar spondylitis due to Pseudomonas aeruginosa. An overshooting of Th1 expression, such as tumor necrosis factor alpha, bumped up by his uremia as a result of P. aeruginosa infection may hasten SCCA. Therefore, SCCA might additionally serve as a serological marker for infection besides squamous cell cancer, and its false-positive increase also highlights the appropriateness of tumor marker screening.

The increase of blood anticardiolipin antibody depends on the underlying etiology in cerebral ischemia.

Although anticardiolipin antibody (aCL) has been suggested to be a potent risk factor for thrombosis and atherosclerosis in multiple arterial beds, conflicting results exist between aCL and cerebral ischemia in the general stroke population. To elucidate if this discrepancy relates to the heterogeneity of underlying etiologies, the blood beta(2)-glycoprotein I dependent-aCL in 432 Taiwanese adults was examined. The associated cerebral ischemia in these patients was classified into five subtypes according to the cause of cerebral ischemia. The results were compared with those in 100 healthy controls. A definite increase of aCL-IgG isotype was found in 41 patients (9.35%) and four controls (4.0%). The relative risk was 2.52. The frequency of increased aCL-IgG was 12.2%, 12.8%, 8.8%, 3.9%, and 3.5% in patients with large-artery atherosclerotic disease, stroke of unknown etiology, small-artery occlusive disease, cardioembolism, and stroke of other known etiology, respectively. Only patients with large-artery atherosclerotic disease (p<0.025) and stroke of unknown etiology (p<0.05) had higher frequencies of increased aCL than those in control subjects. The frequencies of abnormal results of activated partial thromboplastin time, antinuclear factor, Coombs' test, and venereal disease research laboratory were 2.84%, 1.22%, 1.02%, and 1.34% in these 41 patients, respectively. Accordingly, aCL-IgG selectively increases in patients with large-artery atherosclerosis and stroke of unknown etiology, reflecting selective activation of humoral immunity for aCL in the pathogenesis of cerebral ischemia.

Diabetic inferior division palsy of the oculomotor nerve.

In this case report we present a diabetic patient with isolated, pupil-sparing, incomplete inferior division third cranial nerve palsy. Laboratory tests for the evaluation of thyroid function, infection, myasthenia gravis and autoimmune diseases were normal. Cranial computed tomography, magnetic resonance imaging and magnetic resonance angiography also showed normal findings. Accordingly, diabetes related vasculopathic third nerve palsy was suggested. The ocular signs of oculomotor palsy completely disappeared 2 months later. Although this clinical entity is rarely reported, differential diagnosis with pupil-sparing third nerve palsy of other etiologies such as compression by an aneurysm or tumor still need to be investigated.

Tracheostomal fire during an elective tracheostomy.

Some surgeries, a tracheostomy and oral surgeries in particular, have a higher risk of fire. Often it can occur when leaking flammable gas contacts an ignition heat source. Fire during a tracheostomy can sometimes be seen to produce different degrees of insult to the patient. The essential components of a fire, i.e., the fuel source, ignition, and an oxidizer, can be avoided or blocked in order to prevent fires from occurring. Herein, we discuss a fire during a tracheotomy, and ways to avoid its occurrence, and how to stop once it gets started. This case demonstrates 2 important points regarding tracheostomies. First, extreme caution should be exercised when cautery is used in an approximately 100% oxygen gaseous condition, and second, immediate extubation is not absolutely necessary if a fire breaks out during a tracheostomy. The procedures which can be taken when facing this type of emergency are also considered and discussed.

The increase of blood anticardiolipin antibody depends on the underlying etiology in cerebral ischemia.

Although anticardiolipin antibody (aCL) has been suggested to be a potent risk factor for thrombosis and atherosclerosis in multiple arterial beds, conflicting results still exist between aCL and cerebral ischemia in the general stroke population. To elucidate if this discrepancy relates to the heterogeneity of underlying etiologies, blood beta(2)-glycoprotein I dependent-aCL was evaluated in 432 Taiwanese adults associated with cerebral ischemia who were classified into five subtypes according to their causes of cerebral ischemia. The results were compared with those in 100 healthy controls. A definite increase of aCL-IgG isotype was found in 41 patients (9.35%) and four controls (4.0%). The relative risk was 2.52. The frequency of increased aCL-IgG was 12.2%, 12.8%, 8.8%, 3.9%, and 3.5% in patients with large-artery atherosclerotic disease, stroke of unknown etiology, small-artery occlusive disease, cardioembolism, and stroke of other known etiology, respectively. Only patient with large-artery atherosclerotic disease (p<0.025) and stroke of unknown etiology (p<0.05) had a higher frequency of increased aCL than control. The frequencies of abnormal result of activated partial thromboplastin time, antinuclear factor, Coombs' test, and venereal disease research laboratory were 2.84%, 1.22%, 1.02%, and 1.34% in these 41 patients, respectively. Accordingly, aCL-IgG selectively increases in patients with large-artery atherosclerosis and stroke of unknown etiology, reflecting selective activation of humoral immunity for aCL in the pathogenesis of cerebral ischemia.

Intranasal tooth: report of three cases.

Intranasal teeth are uncommon, with only a few reported cases in the past few decades. The clinical manifestations of an intranasal tooth are quite variable. Unilateral nasal obstruction is a common complaint, but even though nasal symptoms are present, an intranasal tooth can be an incidental finding during routine examination in patients without nasal discomfort. Although the diagnosis is not difficult to make, a complete workup that included radiological investigations is important before any surgery is attempted. Transnasal endoscopic surgical approaches have been described with no evidence of recurrence or complications in similar cases. Herein, three patients with an intranasal tooth are described, along with possible etiologies, potential complications, differential diagnoses and their treatments.

Paraganglioma presenting as chronic otitis media with cholesteatoma: pitfalls and strategies.

The coexistence of paraganglioma with cholesteatoma is a very rare clinical disorder. The clinical presentations are nonspecific. There may be radiological characteristics of either cholesteatoma or paraganglioma in the middle ear area, but the diagnosis of the coexistence of the 2 is usually made only postoperatively. Here is such a case that is made more interesting not only because it initially presented with conductive hearing loss but also because the clinical picture mimicked chronic otitis media. The patient underwent postauricular tympanomastoidectomy with extended facial recess approach to remove the tumor. No evidence of recurrence and complications were noted.

Serial changes in platelet activation in patients after ischemic stroke: role of pharmacodynamic modulation.

BACKGROUND AND PURPOSE: Enhanced platelet activity has previously been reported in the acute phase after ischemic stroke. We tested the hypothesis that activated platelets (expressed by CD62p) are substantially increased in the acute stage after a stroke and decrease thereafter, and that antiplatelet therapies can suppress CD62p expression. METHODS: We serially examined platelet CD62p expression using flow cytometry after acute ischemic stroke in 87 consecutive patients. The CD62p expression was also evaluated in 20 healthy volunteers and 33 at-risk control subjects. RESULTS: CD62p expression was significantly higher in the acute phase after ischemic stroke than in normal and at-risk control subjects (both P<0.0001). CD62p expression decreased to a significantly lower level on day 21, and to a substantially lower level on day 90. CD62p expression was not significantly suppressed by warfarin. However, CD62p expression was significantly suppressed by aspirin treatment (P=0.024) and more substantially suppressed by clopidogrel (P<0.0001) on day 90. Furthermore, only clopidogrel treatment (P=0.0016) was significantly independently associated with decreased CD62p expression on day 90. CONCLUSIONS: Platelet activation was significantly increased in acute ischemic stroke and substantially decreased thereafter. The lesser long-term pharmacodynamic potency of aspirin relative to clopidogrel raises the prospect of the need for more effective antiplatelet agents or a synergistic combination therapy for stroke prevention in the future.