EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart

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Biochemical analysis of two cell surface glycoprotein complexes, very common antigen 1 and very common antigen 2. Relationship to very late activation T cell antigens

Two mouse monoclonal antibodies (mAb), AJ2 and J143, define two related human cell surface protein complexes, very common antigen 1 (VCA-1) and very common antigen 2 (VCA-2). In the present report, these complexes have been defined with respect to: (i) subunit arrangement; (ii) monoclonal antibody binding sites; (iii) carbohydrate content; (iv) homology to other cell surface protein complexes; and (v) possible functional roles. Analysis of the antigens from a human melanoma cell line, MeWo, reveals that VCA-1 is a noncovalently linked heterodimer of 170- and 140 (designated 1401)-kDa polypeptides. mAb AJ2 reacts with an epitope on the 1401-kDa polypeptide. VCA-2 is composed of the same 1401-kDa polypeptide as VCA-1 and another 170-kDa species; this 170-kDa species consists of a second distinct 140-kDa (designated 140(2)) and a 30-kDa polypeptide which are disulfide-bonded. Indirect evidence indicates that mAb J143 reacts with an epitope on this 170-kDa complex. Peptide mapping has shown that the complexes are members of a family of cell surface proteins that include antigens present on activated T cells (designated very late activation antigens). Since VCA-2 is found predominantly on the basal membrane of adherent cells and its expression increases 12-fold when HUT-102 lymphoblastoid cells are grown as an adherent cell culture, we suggest that VCA-2 plays a role in cellular adherence.     

Analysis of Adenosine Immunoreactivity, Uptake, and Release in Purified Cultures of Developing Chick Embryo Retinal Neurons and Photoreceptors

We have investigated the presence of endogenous adenosine and of mechanisms for adenosine uptake and release in chick embryo retinal neurons and photoreceptors grown in purified cultures in the absence of glial cells. Simultaneous autoradiographic and immunocytochemical analysis showed that endogenous adenosine and the uptake mechanism for this nucleoside colocalize in practically all the photoreceptors, but only in approximately 20% of the neurons. Approximately 25% of the neurons showed either immunocytochemical labeling or autoradiographic labeling, while greater than 50% of the neurons were unlabeled with both techniques. [3H]Adenosine uptake was saturable and could be inhibited by nitrobenzylthioinosine and dipyridamole and by pretreatment of the [3H]adenosine with adenosine deaminase. Although these observations indicate that the uptake is specific for adenosine, only 35% of accumulated radioactivity was associated with adenosine, with the remaining 65% representing inosine, hypoxanthine, and nucleotides plus uric acid. Adenosine as well as several of its metabolites were released by the cells under basal as well as K(+)-stimulated conditions. Potassium-enhanced release was blocked by 10 mM CoCl2 or in Ca2(+)-free, Mg2(+)-rich solutions. The results indicate that retinal cells that synthesize, store, and release adenosine differentiate early during embryogenesis and are therefore consistent with a hypothetical role for adenosine in retinal development.     

Cytokeratin expression in human respiratory epithelium of nasal polyps and turbinates

The cytokertatins in respiratory epithelial cells (REC) of human nasal polyps and turbinates were analyzed by immunohistochemistry. Cytokeratin 19 (CK19) was present in all REC, CK5 and 14 were expressed primarily in basal cells, and CK7, 8, and 18 were found in suprabasal cells. Differences in cytoplasmic locations were also apparent among the individual cytokeratins. CK13 was not detected in any REC of these tissues. The results indicate the profile of cytokeratins in REC of human nasal polyps and turbinates is essentially identical to that of REC in the more distal respiratory tract.     

Occurrence of subcutaneous zygomycosis (Entomophthoromycosis Basidiobolae) caused by Basidiobolus haptosporus with pulmonary involvement

A case of a two-year-old boy with multiple subcutaneous lesions caused by Basidiobolus haptosporus is presented.The child had also a non-toxic familial goiter and clinical and radiological features of a pulmonary illness. The pulmonary manifestations only disappeared with the treatment with potassium iodide. The authors think that the pulmonary lesions must have arisen by direct spread of the fungus from the subcutaneous lesions of the chest.     

Isolation and characterization of the Mg2(+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations

Preparations of sarcoplasmic reticulum vesicles, obtained according to the method of Eletr and Inesi (Biochim. Biophys. Acta (1972) 282, 174), contained both Mg2(+)-ATPase and Ca2+, Mg2(+)-ATPase activity. The two enzymes were solubilized by a mixture of digitonin and lysophosphatidylcholine and separated on a DEAE-cellulose column eluted with a discontinuous gradient of NaCl. The Mg2(+)-ATPase activity was eluted with 0.43 M NaCl. The Ca2+,Mg2(+)-ATPase was obtained by increasing the NaCl concentration of the elution medium to 0.40 M. The fraction eluted with 0.043 M NaCl was insensitive to micromolar concentrations of calcium, resistant to oligomycin, ouabain, orthovanadate and thiocyanate, and was inhibited by low concentrations of Triton X-100. The enzyme showed a single apparent Km for MgATP in the range of 0.2 mM and a Vm of 2.9 mumol Pi.min-1.mg-1 protein. Activity was maximal over a broad peak between pH 6.0-8.0. Hydrolysis of ATP was unaffected by dimethylsulfoxide concentrations up to 20% (v/v) and was inhibited at higher concentrations. The enzyme was not phosphorylated by either 32Pi or [gamma-32P]ATP at significant levels when compared with the Ca2+,Mg2(+)-ATPase in an EGTA-containing medium. The kinetic pattern of the Mg2(+)-ATPase was distinctly different from that of the Ca2+,Mg2(+)-ATPase under the same conditions. The fraction eluted from the DEAE-cellulose column was subjected to electrophoresis under non-denaturing conditions. Only one band with Mg2(+)-ATPase activity was detected. The Mg2(+)-ATPase migrated much slower than the Ca2+,Mg2(+)-ATPase under non-denaturing conditions, whereas both enzymes had a molecular mass of 105 kDa on SDS gel electrophoresis.     

Meta-omics analysis indicates the saliva microbiome and its proteins associated with the prognosis of oral cancer patients

Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.     

P-I Snake Venom Metalloproteinase Is Able to Activate the Complement System by Direct Cleavage of Central Components of the Cascade

Background

Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom.

Results

Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity.

Conclusion

We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.     

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.