Deoxyribonucleic acid hybridization among strains of lactobacilli

Hybridization of deoxyribonucleic acid (DNA) from Lactobacillus bulgaricus (ATCC 11842) with DNA of L. lactis (ATCC 12315), L. helveticus (ATCC 15009), and L. jugurt (ATCC 521) showed 86.0% reassociation with L. lactis, 4.8% with L. helveticus, and none with L. jugurt.     

Expression of epidermal growth factor in the rat kidney

Summary The renal localization and the site of synthesis of epidermal growth factor (EGF) were investigated in the rat kidney by immunohistochemistry and in situ hybridization techniques. EGF was localized in the cells of the thick ascending limb of Henle (TAL) and distal convoluted tubule (DCT). At the ultrastructural level, EGF immunoreactivity was distributed on the apical membrane and trans-Golgi complex of the TAL and DCT cells. These segments of the rat nephron also hybridized to prepro-EGF cRNA probes in a specific manner, indicating that TAL and DCT are the sites of EGF synthesis in the rat kidney.     

Regulation of lipogenesis in isolated hepatocytes by triglyceride-rich lipoproteins.

Very low density lipoproteins, chylomicrons, and remnants caused, within an hour, significant inhibition of fatty acid synthesis but not cholesterol synthesis in hepatocytes isolated from meal-fed rats. In contrast, low density lipoproteins, high density lipoproteins, and the serum fraction of density greater than 1.21 failed to significantly inhibit either fatty acid or cholesterol synthesis within 1 h. The Scatchard plots of specific binding showed that rat and human very low density lipoproteins interact with the high affinity sites on the hepatocytes with the apparent dissociation constants of 64 and 106 nM, respectively. These data also indicated that each hepatocyte was capable of binding 6 X 10(5) molecules of very low density lipoproteins.     

Rat brain myo-inositol 3-phosphate synthase is a phosphoprotein

The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S₅₂₄), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S₅₂₄, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S₅₂₄, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S₅₂₄ and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.     

Uptake of glucose and orthophosphate by the american oyster.

The uptake, by oysters, of glucose is approximately 10–15 ng/hr/g wet weight, when glucose concentration in the saline was at 50 μg/ml. The removal of inorganic orthophosphate was approximately at a rate of 0.36 ng/hr/g wet weight. Radioactive studies indicate that these substrates are metabolized by oysters. The technique developed can be used to study oyster metabolism in the laboratory.     

Micronucleation by mitosis inhibitors in developing microspores of Spathiphyllum wallisii Regel

Key message

We developed an efficient protocol for chromosome scattering in Spathiphyllum microspores. The effects of plant material, developmental age, genotype and antimicrotubular toxin type, exposure and concentration were evaluated.

Abstract

Asymmetric hybridization through microprotoplast-mediated chromosome transfer (MMCT) is a known method for overcoming sexual breeding barriers between distantly related plant species. To obtain microprotoplasts, it is necessary to induce mass micronucleation either in somatic or gametic cells. We have tested the efficiency for micronuclei induction of five mitosis inhibitors, amiprophos-methyl (APM), butamiphos (BUT), chlorpropham (CIPC), oryzalin (ORY) and propyzamide (PRO), on developing microspores of diploid Spathiphyllum wallisii Regel. Besides the used toxins, also the effect of their concentrations and incubation period as well as plant genotypes and material was tested. We observed micronuclei (MNi) in pollen mother cells, dyads and tetrads as well as other abnormalities such as ball metaphases and chromosome bridges. The flower position on the spadix and the type of starting material (dissected anthers vs. complete spadices) did not significantly influence micronucleation frequencies. The highest micronucleation index of 86 % was obtained in microspores treated with 10 μM ORY during 72 h. All six genotypes tested formed micronuclei after this particular treatment, although the efficiency varied between cultivars. Next to ORY, CIPC was also a very efficient MNi inducer. The average number of MNi found in micronucleated cells varied between 1.67–6.44 for CIPC and 0.83–5.50 for ORY. The maximal number of MNi observed was 12 for CIPC and 9 for ORY. Our results demonstrate that CIPC and ORY can be applied for mass micronucleation on developing microspores of S. wallisii as a first step of MMCT in aroid interspecific or intergeneric breeding.     

Statistical Analysis of Peptide-Induced Graded and All-or-None Fluxes in?Giant Vesicles

Antimicrobial, cytolytic, and cell-penetrating peptides induce pores or perturbations in phospholipid membranes that result in fluxes of dyes into or out of lipid vesicles. Here we examine the fluxes induced by four of these membrane-active peptides in giant unilamellar vesicles. The type of flux is determined from the modality of the distributions of vesicles as a function of their dye content using the statistical Hartigan dip test. Graded and all-or-none fluxes correspond to unimodal and bimodal distributions, respectively. To understand how these distributions arise, we perform Monte Carlo simulations of peptide-induced dye flux into vesicles using a very simple model. The modality of the distributions depends on the rate constants of pore opening and closing, and dye flux. If the rate constants of pore opening and closing are both much smaller than that of dye flux through the pore, all-or-none influx occurs. However, if one of them, especially the rate constant for pore opening, increases significantly relative to the flux rate constant, the process becomes graded. In the experiments, we find that the flux type is the same in giant and large vesicles, for all peptides except one. But this one exception indicates that the flux type cannot be used to unambiguously predict the mechanism of membrane permeabilization by the peptides.     

Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis.     

Surface loop dynamics in adeno-associated virus capsid assembly

The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.     

Biological evaluation,molecular docking and DNA interaction studies of coordination compounds gleaned from a pyrazolone incorporated ligand

Abstract

In this work, we have synthesized a few novel mononuclear complexes of Cu(II), Co(II), Ni(II) and Zn(II) using a pyrazolone-derived Schiff base ligand. They were characterized by spectroscopic and analytical methods. The elemental analyses, UV-Vis, magnetic moment values and molar conductance of the complexes reveal that the complexes adopt an octahedral arrangement around the central metal ions. The interaction of complexes with CT-DNA was studied by absorption spectral titration and viscosity measurements. The observed data show that the complexes bind with CT-DNA via an intercalation mode. Efficient pUC18 DNA cleavage ability of the synthesized compounds was explored by gel electrophoresis. The antimicrobial activity of these compounds against a set of bacterial and fungal strains reveals that the complexes exhibit better activity than the free ligand. Moreover, all the complexes were evaluated against two cancer (HeLa and HepG2) and one normal (NHDF) cell lines. The data were compared with cisplatin. Anti–inflammatory activity has been experimentally validated which proves that theoretical predictions concur with the experimental results. In addition, molecular docking studies have been performed to consider the nature of binding mode and binding affinity of these compounds with DNA (1BNA) and protein (3hb5). These studies reveal that the mode of binding is intercalation and the complexes have higher binding energy scores than the free ligand.