Highly repetitive sequences in B chromosomes of Secale cereale revealed by fluorescence in situ hybridization.

An analysis of the presence and distribution of the rye and wheat repeated sequences in rye B chromosomes was carried out by fluorescent in situ hybridization. Probes used consisted of three highly repetitive sequences from rye (pSc119.2, pSc74, and pSc34) and the multigene families for the 25S-5.8S-18S and 5S rDNA from wheat (pTa71 and pTa794, respectively). pSc74 and pSc119.2 showed hybridization signals in the telomeric regions of rye B chromosomes. The remaining DNA clones did not hybridize to the B chromosomes.     

Schizophyllan (SPG)-treated macrophages and anti-tumor activities against syngeneic and allogeneic tumor cells

We tested anti-tumor activities of macrophages treated with a neutral polysaccharide, schizophyllan (SPG), against syngeneic and allogeneic tumor cell lines. SPG was a macrophage stimulant which was not mitogenic to lymphocytes. That made a sharp contrast with the data that Corynebacterium parvum, BCG, and muramyl dipeptide (MDF) were macrophage stimulants which had lymphocyte-activating properties. Treatment of SPG-treated PEC with Thy12 monoclonal antibody and guinea pig complement did not affect the capabilities of tumor-cell-growth suppression by the treated PEC. Thus, the effector cells were peritoneal adherent cells (macrophages morphologically) and effector-to-target contact seemed to be necessary for effective tumor-cell-growth inhibition, although contradictory data exist for this. Murine peritoneal adherent cells harvested 4 days after a single IP injection of SPG at a dose of 100 mg/kg body weight of mouse showed the most prominent cytostatic and cytotoxic activities against syngeneic and allogeneic tumor cells. The distribution of anti-tumor activity in macrophages of various sizes followed the same pattern as macrophages treated with C. Parvum, i.e., larger macrophages showed more remarkable anti-tumor activity. Crude nonadherent peritoneal cells incubated with SPG at a concentration of 10 micrograms/ml, 100 micrograms/ml, or 1 mg/ml did not secrete lymphokine that rendered macrophages cytotoxic, while ConA-treated nonadherent cells did so. Furthermore, spleen cells treated with SPG in vivo did not secrete macrophage-activating lymphokine in the presence of SPG. On the other hand, addition of 1 mg/ml of SPG-treated peritoneal adherent cells and bone-marrow-derived macrophages in vitro rendered them cytotoxic to a moderate degree. This implies that SPG may activate macrophages directly, allowing them to become cytotoxic in the peritoneal cavity. Lastly, SPG could induce production of II-1-like factor to a moderate degree. SPG, whose molecular structure is well elucidated, will provide us with a strong tool to analyze the mechanism of macrophage activation both in vitro and in vivo.     

A study of the binding of Mn2+ to bovine pancreatic deoxyribonuclease I and to deoxyribonucleic acid by electron paramagnetic resonance.

EPR studies of Mn2+ binding to bovine pancreatic deoxyribonuclease I show that the enzyme can bind three Mn2+ ions at pH 7.5 and 2 degrees. Two sites bind Mn2+ strongly, with a Kd of 10(-4)M, and the third binds Mn2+ weakly, with a Kd of 10(-3)M. Ca2+ competes with the two strong sites, whereas Mg2+ competes only with one of them, indicating that both sites are not equivalent. Mn2+ binding to DNA has been confirmed by EPR measurements. Two types of sites, with different affinities for Mn2+ binding, were found on DNA molecules, one with a Kd of 1.2 times 10(-4)M and the other with a Kd of 10(-3)M. Mg2+ ions can displace Mn2+ from the high affinity sites, but not from the low affinity sites. These results suggest the Mn2+ binds not only to the phosphate groups, but also to the electron donor groups of the base rings.     

Characterization of a xylose containing oligosaccharide,an inhibitor of multidrug resistance in Staphylococcus aureus,from Ipomoea pes-caprae

Pescaprein XVIII (1), a type of bacterial efflux pump inhibitor, was obtained from the CHCl₃-soluble resin glycosides of beach morning glory (Ipomoea pes-caprae). The glycosidation sequence for pescaproside C, the glycosidic acid core of the lipophilic macrolactone 1 containing d-xylose and l-rhamnose, was characterized by means of several NMR techniques and FAB mass spectrometry. Recycling HPLC also yielded eight non-cytotoxic bacterial resistance modifiers, the two pescapreins XIX (2) and XX (3) as well as the known murucoidin VI (4), pecapreins II (6) and III (7), and stoloniferins III (5), IX (8) and X (9), all of which contain simonic acid B as their oligosaccharide core. Compounds 1–9 were tested for in vitro antibacterial and resistance-modifying activity against strains of Staphylococcus aureus possessing multidrug resistance efflux mechanisms. All of the pescapreins potentiated the action of norfloxacin against the NorA over-expressing strain by 4-fold (8 μg/mL from 32 μg/mL) at a concentration of 25 μg/mL.     

Native and heterologous production of bacteriocins from gram-positive microorganisms

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.     

The Effects of Acetylcholine on the Turnover of Phosphatidic Acid and Phosphoinositide in Sympathetic Ganglia, and in Various Parts of the Central Nervous System in Vitro

The effect of acetylcholine on the incorporation of P³² into the individual phosphatides in slices of various structures of the nervous system has been studied. There was a marked stimulation of P³² incorporation into phosphoinositide and phosphatidic acid, but not into phosphatidyl choline and phosphatidyl ethanolamine, in the cat stellate and celiac ganglia in vitro. Acetylcholine stimulated P³² incorporation into certain phosphatides, primarily phosphoinositide and phosphatidic acid, in several structures of the cat and guinea pig brain; there was little or no effect of acetylcholine on phosphatide turnover in the inferior corpora quadrigsemina and cerebellar cortex. The suggestion is made that the phospholipid effect can best be explained as being concerned with the active transport of sodium ions out of the cell across the postsynaptic membrane of cholinergic neurons in response to acetylcholine.     

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.     

Revising the Absolute Configurations of Coatlines via Density Functional Theory Calculations of Electronic Circular Dichroism Spectra

Coatline A ( 1 ) and α‐epi‐coatline A ( 4 ) co‐occur in the trunk extract of Andira coriacea. Inspection of their chiroptical properties led to intriguing results. After a careful examination of the experimental data used for the previously reported absolute configuration of these compounds, some uncertainties were identified. A combined theoretical approach including conformational analyses and calculation of electronic circular dichroism (ECD) spectra, in addition with experimental data obtained for schoepfin A ( 5 ) and the new schoepfin D ( 6 ) isolated from Senna quinquangulata, allowed the revision of the absolute configuration of coatlines A ( 1 ) and B ( 2 ). Chirality 25:180–184, 2013. © 2012 Wiley Periodicals, Inc.