Electrophoretic resolution and fluorescence detection of N-linked glycoprotein oligosaccharides after reductive amination with 8-aminonaphthalene-1,3,6-trisulphonic acid.

The relative mobilities of various N-linked oligosaccharides reductively aminated to the charged fluorophore 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS) were determined by electrophoresis on high-density polyacrylamide slab gels. Each ANTS-derivatized oligosaccharide was assigned a relative migration index (RMI) expressed in terms of glucose equivalents, which was conveniently estimated by reference to a homologous series of ANTS--maltooligosaccharides run on each gel as oligosaccharide size standards. High-mannose-, complex- and hybrid-type structures were generally well resolved and easily visualized at picomole levels by simple UV light excitation. Application of these methods for the qualitative analysis of the oligosaccharides released from bovine fetuin and bovine asialofetuin by peptide-N-glycosidase F illustrates the usefulness of these techniques as fast, simple, and inexpensive tools for the characterization of N-linked oligosaccharides attached to glycoproteins.     

Plasmin binding to the plasminogen receptor enhances catalytic efficiency and activates the receptor for subsequent ligand binding.

Specific cell surface receptors for plasminogen (Pg) are expressed by a wide variety of cell types. The colocalization of receptors for Pg and its activators restricts plasmin (Pm) activity to specific sites and serves to promote fibrinolysis and local Pg activation. These studies show that both Pg and Pm bind to cellular receptors on monocytoid U937 cells. Limited Pm pretreatment of the cells enhances total Pg binding and alters the kinetics of Pm binding. Furthermore, surface-bound Pg is converted to Pm in the absence of exogenous activators. Cell-bound Pm exhibits a 12-fold increase in catalytic efficiency (kcat/Km) relative to Pm free in solution. These studies demonstrate that Pg/Pm receptor occupancy can be regulated by Pm in the microenvironment and may play a significant regulatory role in fibrinolysis and extravascular proteolysis.     

Relative mitogenic activities of various estrogens and antiestrogens

The abilities of a variety of estrogens and antiestrogens to stimulate DNA synthesis in the prepuberal rat uterus were compared. One microgram of each compound was administered in vivo via a single intraperitoneal injection. DNA synthesis was assayed in vitro in isolated nuclei 24 h later. The relative mitogenicities of the steroidal estrogens were: 16 alpha-E2 less than 17 alpha-E2 = E3 = 16-EpiE3 less than 16 beta-E2 = 17 beta-E2. The potencies of several nonsteroidal estrogens were also tested. Indenestrol A was as potent at 17 beta-E2, whereas indanestrol and dimethylstilbestrol had weaker activities. The antiestrogens, nafoxidine and 4-hydroxytamoxifen, were both potent stimulators of DNA synthesis. The abilities of an estrogen to stimulate increases in uterine wet weight, DNA polymerase alpha activities, and DNA synthesis in uterine nuclei 24 h after injection were closely correlated. Because the magnitude of the stimulation of DNA synthesis was greatest, its measurement is the most sensitive of these assays of uterotrophic activity.     

VII Convegno Parco Naturale di Cavriglia 27–29 ottobre 1989 Abstracts

VII Convegno Parco Naturale di Cavriglia 27–29 ottobre 1989 Abstracts     

Night roosting and the nocturnal time budget of the little brown bat,Myotis lucifugus: Effects of reproductive status,prey density,and environmental conditions

Summary The insectivorous bat Myotis lucifugus typically apportions the night into two foraging periods separated by an interval of night roosting. During this interval, many bats occupy roosts that are used exclusively at night and are spatially separate from maternity roosts. The proportion of the night which bats spend roosting, and thus the proportion spent foraging, vary both daily and seasonally in relation to the reproductive condition of the bats, prey density, and ambient temperature. A single, continuous night roosting period is observed during pregnancy. During lactation, females return to maternity roosts between foraging bouts, and night roosts are used only briefly and sporadically. Maximum use of night roosts occurs in late summer after young become volant. Superimposed upon these seasonal trends is day-to-day variation in the bats' nightly time budget. Long night roosting periods and short foraging periods are associated with cool nights and low prey density. This behavioral response may minimize energetic losses during periods of food scarcity.     

Taxonomic relationships among strains of the anaerobic bacterium Bacteroides ruminicola determined by DNA and extracellular polysaccharide analysis.

DNA and extracellular polysaccharide (EPS) analyses were performed on 14 strains of Bacteroides ruminicola. The guanine-plus-cytosine (G+C) base contents, determined from the buoyant densities of chromosomal DNAs, showed a broad range of values, from 37.6 to 50.9 mol%. DNA hybridization showed generally low DNA relatedness among the strains. Seven strains formed two groups of closely related bacteria consisting of five (group 1) and two (group 2) strains, and another strain, E42g, showed moderate relatedness to group 1 strains. However, the remaining six strains were not related to any of the other strains. DNA reassociation indicates that the strains constitute a genetically diverse group representing as many as nine separate species. EPS analysis showed that the strains produced EPS with rather uniform sugar compositions, which did not correlate with strain relationships determined by DNA analysis. Four strains had EPS with acidic sugars or unknown compounds. The EPS of strain 20-63 contained the unusual acidic sugar 4-O-(1-carboxyethyl)-rhamnose. This monosaccharide has been shown to occur in nature in only one other bacterial species.     

Antimutagenicity profiles for some model compounds

The concept of activity profile listings and plots, already applied successfully to the display of mutagenicity data, has been modified for application to antimutagenicity data. The activity profiles are bar graphs that have been organized in two general ways: for antimutagens that have been tested in combination with a given mutagen and for mutagens that have been tested in combination with a given antimutagen. Doses from both the mutagen and the antimutagen are displayed and plotted together with results on enhancement or inhibition of mutagenic activity. The short-term tests that have been used extensively to identify mutagens and potential carcinogens are increasingly being used to identify antimutagens and potential anticarcinogens. Three model mutagens, N-methyl-N'-nitro-N-nitrosoguanidine, aflatoxin B1 and benzo[a]pyrene, and 4 model antimutagens, butylated hydroxyanisole, butylated hydroxytoluene, glutathione and disulfiram, were selected from the data surveyed in the published literature. It is not clear at the present time whether the inhibition of carcinogen-induced mutation is a good indicator of anticarcinogenic properties, and further research is needed. Nevertheless, the activity profiles are useful for the assessment of the available antimutagenesis data by providing rapid visualization of considerable dose information and experimental results.     

Lysophosphatidic acid down-regulates stress fibers and up-regulates pro-matrix metalloproteinase-2 activation in ovarian cancer cells

Epithelial ovarian cancer (EOC) is asymptomatic at early stages and is often diagnosed late when tumor cells are highly metastatic. Lysophosphatidic acid (LPA) has been implicated in ovarian oncogenesis as levels of this lipid are elevated in patient ascites and plasma. Because the underlying mechanism governing LPA regulation of matrix metalloproteinase-2 (MMP-2) activation remains undefined, we investigated the relationship between LPA-induced changes in actin microfilament organization and MMP-2 enzymatic activity. We report that when cells were cultured at a high density, LPA mediated stress fiber and focal adhesion disassembly and significantly repressed RhoA activity in EOC cells. Inhibition of Rho-kinase/ROCK enhanced both LPA-stimulated loss of stress fibers and pro-MMP-2 activation. In contrast, expression of the constitutively active RhoA(G14V) mutant diminished LPA-induced pro-MMP-2 activation. LPA had no effects on membrane type 1-MMP or tissue inhibitor of metalloproteinase-2 expression, but up-regulated MMP-2 levels, contributing to the induction of MMP-2 activation. Interestingly, when cells were cultured at a low density, stress fibers were present after LPA stimulation, and ROCK activity was required for EOC cell migration. Collectively, these results were consistent with a model in which LPA stimulates the metastatic dissemination of EOC cells by initiating loss of adhesion and metalloproteinase activation.     

Serological responses to human virome define clinical outcomes of Italian patients infected with SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic respiratory infectious disease COVID-19. However, clinical manifestations and outcomes differ significantly among COVID-19 patients, ranging from asymptomatic to extremely severe, and it remains unclear what drives these disparities. Here, we studied 159 sequentially enrolled hospitalized patients with COVID-19-associated pneumonia from Brescia, Italy using the VirScan phage-display method to characterize circulating antibodies binding to 96,179 viral peptides encoded by 1,276 strains of human viruses. SARS-CoV-2 infection was associated with a marked increase in immune antibody repertoires against many known pathogenic and non-pathogenic human viruses. This antiviral antibody response was linked to longitudinal trajectories of disease severity and was further confirmed in additional 125 COVID-19 patients from the same geographical region in Northern Italy. By applying a machine-learning-based strategy, a viral exposure signature predictive of COVID-19-related disease severity linked to patient survival was developed and validated. These results provide a basis for understanding the role of memory B-cell repertoire to viral epitopes in COVID-19-related symptoms and suggest that a unique anti-viral antibody repertoire signature may be useful to define COVID-19 clinical severity.