“Hypermethylation” of anthranilic acid-labeled sugars confers the selectivity required for liquid chromatography-mass spectrometry

Analysis of the monosaccharides of complex carbohydrates is often performed by liquid chromatography with fluorescence detection. Unfortunately, methylated sugars, unusual amino- or deoxysugars and incomplete hydrolysis can lead to erroneous assignments of peaks. Here, we demonstrate that a volatile buffer system is suitable for the separation of anthranilic acid labeled sugars. It allows off-line examination of peaks by electrospray mass spectrometry. Approaches towards on-line mass spectrometric detection using reversed-phase or porous graphitic carbon columns fell short of achieving sufficient separation of the relevant isobaric sugars. Adequate chromatographic performance for isomeric sugars was achieved with reversed-phase chromatography of “hyper”-methylated anthranilic acid-labeled monosaccharides. Deuteromethyl iodide facilitates the discovery of naturally methylated sugars and identification of their parent monosaccharide as demonstrated with N-glycans of the snail Achatina fulica, where two thirds of the galactoses and a quarter of the mannoses were methylated.     

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15–1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.     

Innate defense against influenza A virus: activity of human neutrophil defensins and interactions of defensins with surfactant protein D

Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some instances when relatively SP-D-resistant strains were treated with SP-D and HNPs. HNPs were found to bind to the neck and/or carbohydrate recognition domain of SP-D. This binding was specific because no, or minimal, binding to other collectins was found. HNPs precipitated SP-D from bronchoalveolar lavage fluid and reduced the antiviral activity of bronchoalveolar lavage fluid. HNP-1 and -2 differed somewhat in their independent antiviral activity and their binding to SP-D. These results are relevant to the early phase of host defense against IAV, and suggest a complex interplay between SP-D and HNPs at sites of active inflammation.     

Generation of high curvature membranes mediated by direct endophilin bilayer interactions

Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143-154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133-141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301-312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33-39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.     

Streptacidiphilus jiangxiensis sp. nov., a novel actinomycete isolated from acidic rhizosphere soil in China

The taxonomic position of three acidophilic actinomycetes isolated from acidic rhizosphere soil was established using a polyphasic approach. The morphological and chemical properties of the isolates were found to be consistent with their assignment to the genus Streptacidiphilus. Almost complete 16S rRNA gene sequences determined for the strains were aligned with corresponding sequences of representatives of the genera Kitasatospora, Streptacidiphilus and Streptomyces and phylogenetic trees inferred using three tree-making algorithms. The organisms formed a distinct subclade within the Streptacidiphilus 16S rRNA gene tree. They also shared nearly identical phenotypic profiles and rep-PCR fingerprint patterns that readily distinguished them from representatives of the established species of Streptacidiphilus. It is evident from the genotypic and phenotypic data that the three isolates form a new species in the genus Streptacidiphilus. The name proposed for this new species is Streptacidiphilus jiangxiensis, the type strain is isolate 33214T (= AS 4.1857T = JCM 12277T).     

Presence of Human Papillomavirus DNA in Malignant Neoplasia and Non-Malignant Breast Disease

Breast cancer is the leading cause of cancer death among women worldwide. Multiple extrinsic and intrinsic factors are associated with this disease’s development. Various research groups worldwide have reported the presence of human papillomavirus (HPV) DNA in samples of malignant breast tumors. Although its role in mammary carcinogenesis is not fully understood, it is known that the HPV genome, once inserted into host cells, has oncogenic capabilities. The present study aimed to detect the presence of HPV DNA in 116 breast tissue biopsies and classify them according to their histology. It was found that 50.9% of the breast biopsies analyzed were malignant neoplasms, of which 74.6% were histologically classified as infiltrating ductal carcinoma. In biopsies with non-malignant breast disease, fibroadenoma was the most common benign neoplasm (39.1%). Detection of HPV DNA was performed through nested PCR using the external primer MY09/11 and the internal primer GP5+/6+. A hybridization assay genotyped HPV. HPV DNA was identified in 20.3% (12/59) of malignant neoplasms and 35% non-malignant breast disease (16/46). It was also detected in 27.3% (3/11) of breast tissue biopsies without alteration. However, there are no statistically significant differences between these groups and the existence of HPV DNA (p = 0.2521). Its presence was more frequent in non-malignant alterations than in malignant neoplasias. The most frequent genotypes in the HPV-positive samples were low-risk (LR) HPV-42 followed by high-risk (HR) HPV-31.     

THE HORMONAL INTEGRATION OF SEXUAL REPRODUCTION IN OEDOGONIUM

Rawitscher -Kunkel , Erika , and L. Machlis . (U. California, Berkeley.) The hormonal integration of sexual reproduction in Oedogonium. Amer. Jour. Bot. 49 (2) : 177–183. Illus. 1962.—Sexual reproduction in a heterothallic, nannandrous species of Oedogonium was investigated cytologically and physiologically. Several new observations are reported. Oogonial mother cells release a substance which attracts androspores to them. The androspores, when attached to the oogonial mother cells, grow in well-defined directions apparently in response to a hormone originating in the oogonial mother cells. An oogonial mother cell divides into an oogonium and a suffultory cell only after the attached androspores complete their development into dwarf males, each bearing an antheridium. Presumably the developing dwarf males provide a chemical stimulus for the division of the oogonial mother cell. During development, the oogonia become enveloped in a massive gel which also encases the antheridia cut off at the apical ends of the dwarf male plants. The gel appears to function as a sperm trap, preventing the dissemination of the sperm into the surrounding liquid. The sperm are attracted to the protoplasmic papilla which briefly protrudes through the oogonial pore indicating the operation of a second chemotactic agent.