Complete subsite mapping of a “loopful” GH19 chitinase from rye seeds based on its crystal structure

AbstractCrystallographic analysis of a mutated form of “loopful” GH19 chitinase from rye seeds a double mutant RSC-c, in which Glu67 and Trp72 are mutated to glutamine and alanine, respectively, (RSC-c-E67Q/W72A) in complex with chitin tetrasaccharide (GlcNAc)4 revealed that the entire substrate-binding cleft was completely occupied with the sugar residues of two (GlcNAc)4 molecules. One (GlcNAc)4 molecule bound to subsites −4 to −1, while the other bound to subsites +1 to +4. Comparisons of the main chain conformation between liganded RSC-c-E67Q/W72A and unliganded wild type RSC-c suggested domain motion essential for catalysis. This is the first report on the complete subsite mapping of GH19 chitinase

Membrane topology analysis of HIV-1 envelope glycoprotein gp41

<p>Abstract</p> <p>Background</p> <p>The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system.</p> <p>Results</p> <p>In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability.</p> <p>Conclusions</p> <p>It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.</p

Electroretinographic assessment of rod- and cone-mediated bipolar cell pathways using flicker stimuli in mice

Mouse full-field electroretinograms (ERGs) are dominated by responses of photoreceptors and depolarizing (ON-) bipolar cells, but not much of hyperpolarizing (OFF-) bipolar cells under conventional recording conditions. Here we investigate a novel ERG protocol in mice for functional assessment of the major ON-and OFF- bipolar cell pathways using flicker stimuli for a high luminance with varying frequency up to 30 Hz. Wild- type (WT) and functionally specific transgenic mice (Cnga3(-/-),no cone photoreceptor function;rho(-/-),no rod photoreceptor function;mGluR6(-/-),no ON-bipolar cell function) were examined. The Cnga3(-/-) flicker ERG was similar to the WT flicker ERG at very low stimulus frequencies, whereas ERGs were comparable between WT and rho(-/-) mice at 5 Hz and above. Between 5 and 15 Hz, ERGs in mGluR6(-/-) mice differed in configuration and amplitude from those in WT and rho(-/-) mice;in contrast, response amplitudes above 15 Hz were comparable among WT, rho(-/-) and mGluR6(-/-) mice. In summary, we found three frequency ranges with these conditions that are dominated by activity in the rod pathways (below 5 Hz),cone ON-pathway (between 5 and 15 Hz),and cone OFF-pathway (above 15 Hz) that enables a quick overview of the functionality of the major bipolar cell pathways

Hybrid Desmoplastic/Follicular Ameloblastoma of the Mandible: A Case Report and Review of the Literature

Desmoplastic ameloblastoma (DA) is one of the 6 histopathological subtypes of ameloblastoma. Hybrid lesions in which histopathologically conventional ameloblastoma coexists with areas of DA are rare. A 40-year-old male was referred to our hospital complaining of a swelling in the right premolar region of the mandible. A panoramic radiograph showed an area of radiolucency with a well-defined corticated border, whereas computed tomography revealed a unilocular radiolucent lesion and buccal expansion together with cortical perforation. The lesion was treated via enucleation and curettage of the marginal bone and fenestration. A histopathological examination showed a hybrid ameloblastoma with a pronounced desmoplastic pattern and follicular changes. The patient’s postoperative course has been favorable up to now, and no marked changes have been observed. We presented a case of hybrid ameloblastoma and reviewed the 36 reported cases of hybrid ameloblastoma that have been reported in the English literature

NOS2 polymorphisms associated with the susceptibility to pulmonary arterial hypertension with systemic sclerosis: contribution to the transcriptional activity

Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis. One of several complications of SSc, pulmonary arterial hypertension (PAH) can be refractory to treatment, both novel and established. In the present study we investigated the ratio of circulating nitric oxide to endothelin-1 in patients with both SSc and PAH, and determined whether polymorphisms in NOS2 (the nitric oxide synthase 2 gene) are associated with susceptibility to PAH. Endothelin-1 in plasma and nitric oxide metabolites (nitrate and nitrite) in serum were measured. The nitric oxide/endothelin-1 ratio was significantly lower in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. We confirmed the presence of two single nucleotide polymorphisms at positions -1,026 and -277 and a pentanucleotide repeat (CCTTT) at -2.5 kilobases. There were significant differences in single nucleotide polymorphisms between patients with SSc who had PAH and those who did not, and between patients with both SSc and PAH and healthy control individuals. The CCTTT repeat was significantly shorter in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. Transcriptional activity were analyzed using the luciferase reporter assay. The transcriptional activity of NOS2 was much greater in fibroblasts transfected by a vector with a long allele of the CCTTT repeat than in those transfected by a vector with a short allele. Polymorphisms in the NOS2 gene are associated with transcriptional activity of the NOS2 gene and with susceptibility to SSc-related PAH

Detection of adenovirus hepatitis and acute liver failure in allogeneic hematopoietic stem cell transplant patients

Human adenovirus (HAdV) is an important cause of the common cold and epidemic keratoconjunctivitis in immunocompetent individuals. In immunocompromised patients, HAdV can sometimes cause severe infection such as cystitis, gastroenteritis, pneumonia, encephalitis, hepatitis, or disseminated disease, resulting in significant morbidity and also mortality. In particular, severe cases have been reported in patients after allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Indeed HAdV has been recognized as a pathogen that requires careful monitoring in allo‐HSCT patients. While HAdV hepatitis leading to severe acute liver failure is rare, such liver failure progresses rapidly and is often fatal. Unfortunately, HAdV hepatitis has few characteristic symptoms and physical findings, which makes it difficult to promptly confirm and start treatment. We report here four cases of HAdV hepatitis after allo‐HSCT and their autopsy findings

Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

<p>Abstract</p> <p>Background</p> <p>We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear.</p> <p>Results</p> <p>We examined HIV-1 fusion with a panel of target cells lines and with primary CD4⁺T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells <it>via </it>endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization.</p> <p>Conclusions</p> <p>Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.</p

Acute Common Femoral Artery Occlusion following Total Hip Arthroplasty for an Arthrodesed Hip

Perioperative acute arterial complications occur rarely after total hip arthroplasty (THA) but can be limb or life threatening. Here, we report a case of an arterial thrombosis after primary THA for an arthrodesed hip. The arterial occlusion occurred because of the surgical mobilization against the immovable flexion and adduction position. A review of the literature on vascular complications arising after THA suggests multiple possible mechanisms and clinical presentations that relate to these complications. Specific risk factors can be identified in THAs replacement cases and THA for the arthrodesed hip can be a major risk factor. Most of these vascular complications can be prevented or more efficiently treated by thorough preoperative assessment and careful postoperative monitoring

The critical role of Rap1-GAPs Rasa3 and Sipa1 in T cells for pulmonary transit and egress from the lymph nodes

Rap1-GTPase activates integrins and plays an indispensable role in lymphocyte trafficking, but the importance of Rap1 inactivation in this process remains unknown. Here we identified the Rap1-inactivating proteins Rasa3 and Sipa1 as critical regulators of lymphocyte trafficking. The loss of Rasa3 and Sipa1 in T cells induced spontaneous Rap1 activation and adhesion. As a consequence, T cells deficient in Rasa3 and Sipa1 were trapped in the lung due to firm attachment to capillary beds, while administration of LFA1 antibodies or loss of talin1 or Rap1 rescued lung sequestration. Unexpectedly, mutant T cells exhibited normal extravasation into lymph nodes, fast interstitial migration, even greater chemotactic responses to chemokines and sphingosine-1-phosphate, and entrance into lymphatic sinuses but severely delayed exit: mutant T cells retained high motility in lymphatic sinuses and frequently returned to the lymph node parenchyma, resulting in defective egress. These results reveal the critical trafficking processes that require Rap1 inactivation