Phytoconstituents from the root of Streptocaulon tomentosum and their chemotaxonomical relevance for separation from S. juventas

A new cardenolide, 17β-H-periplogenin-3-O-β-d-digitoxoside (1), and a new pregnane glycoside, Δ⁵-pregnene-3β,16α-diol-d-O-[2,4-O-diacetyl-β-digitalopyranosyl-(1 → 4)-β-d-cymaropyranoside]-16-O-[β-d-glucopyranoside] (2) were isolated from the roots of Streptocaulon tomentosum (Asclepiadaceae) together with a series of known compounds. Their chemotaxonomic significance for the separation of S. tomentosum from Streptocaulon juventas is discussed, suggesting a rather clear distinction of these species.     

Induction of heme oxygenase-1 and heat shock protein 70 in rat hepatocytes: The role of calcium signaling

Stress response genes including heat shock proteins are induced under a variety of conditions to confer cellular protection. This study investigated the role of calcium signaling in the induction of two stress response genes, heme oxygenase-1/hsp32 and hsp70, in isolated rat hepatocytes. Both genes were induced by cellular glutathione depletion. This induction could be inhibited by BAPTA-AM. Culturing in a calcium-free medium prevented the induction of hsp70 gene expression after glutathione depletion without affecting heme oxygenase-1 gene expression. Thapsigargin increased the gene expression of heme oxygenase-1 but not that of hsp70. Thapsigargin-induced heme oxygenase-1 induction was completely inhibited by BAPTA-AM. Incubation with the Ca²⁺-ionophore A23187 augmented heme oxygenase-1 (two-fold) and hsp70 (5.2-fold) mRNA levels. Our data suggests a significant role of Ca²⁺-dependent pathways in the induction of the two stress genes. An increase in the cytoplasmic Ca²⁺ activity seems to play a key role in the cascade of signaling leading to the induction of the two genes. However, the source of Ca²⁺ that fluxes into the cytoplasm seems to be different. Our data provides evidence for a compartmentalization of calcium fluxes, i.e. the Ca²⁺ flux from intracellular stores (e.g. the endoplasmic reticulum) plays a major role in the induction of heme oxygenase-1. By contrast, Ca²⁺ flux from the extracellular medium seems to be a mechanism initiating the cellular signaling cascade leading to hsp70 gene induction.     

Quantitative Superresolution Microscopy Reveals Differences in Nuclear DNA Organization of Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance

The mammalian nucleus has a distinct substructure that cannot be visualized directly by conventional microscopy. In this study, the organization of the DNA within the nucleus of multiple myeloma (MM) cells, their precursor cells (monoclonal gammopathy of undetermined significance; MGUS) and control lymphocytes of the representative patients is visualized and quantified by superresolution microscopy. Three‐dimensional structured illumination microscopy (3D‐SIM) increases the spatial resolution beyond the limits of conventional widefield fluorescence microscopy. 3D‐SIM reveals new insights into the nuclear architecture of cancer as we show for the first time that it resolves organizational differences in intranuclear DNA organization of myeloma cells in MGUS and in MM patients. In addition, we report a significant increase in nuclear submicron DNA structure and structure of the DNA‐free space in myeloma nuclei compared to normal lymphocyte nuclei. Our study provides previously unknown details of the nanoscopic DNA architecture of interphase nuclei of the normal lymphocytes, MGUS and MM cells. This study opens new avenues to understanding the disease progression from MGUS to MM. J. Cell. Biochem. 116: 704–710, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Tumor delivery of ultrasound contrast agents using Shiga toxin B subunit

The present study demonstrates the targeting of ultrasound contrast agents to human xenograft tumors by exploiting the overexpression of the glycolipid Gb3 in neovasculature. To this end, microbubbles were functionalized with a natural Gb3 ligand, the B subunit of the Shiga toxin (STxB). The targeting of Gb3-expressing tumor cells by STxB microbubbles was first shown by flow cytometry and fluorescence microscopy. A significantly higher proportion of STxB microbubbles were associated with Gb3-expressing tumor cells compared to cells in which Gb3 expression was inhibited. Moreover, ultrasonic imaging of culture plates showed a 12 dB contrast enhancement in average backscattered acoustic intensity on the surface of Gb3-expressing cells compared to Gb3-negative cells. Also, a 18 dB contrast enhancement was found in favor of STxB microbubbles compared to unspecific microbubbles. Microbubble signal intensity in subcutaneous tumors in mice was more than twice as high after the injection of STxB-functionalized microbubbles compared to the injection of unspecific microbubbles. These in vitro and in vivo experiments demonstrated that STxB-functionalized microbubbles bind specifically to cells expressing the Gb3 glycolipid. The cell-binding moieties of toxins thus appear as a new group of ligands for angiogenesis imaging with ultrasound.     

Ipangulines and minalobines, chemotaxonomic markers of the infrageneric Ipomoea taxon subgenus Quamoclit, section Mina

A comprehensive GC-MS analysis of 8 Ipomoea species belonging to the subgenus Quamoclit, section Mina revealed that the members of this taxon form combinations of two necine bases with rare necic acids resulting in unique pyrrolizidine alkaloids. The occurrence and diversity of these metabolites show remarkable variations: Some species, especially Ipomoea hederifolia and Ipomoea lobata, are able to synthesize a large number of alkaloids whereas others, especially Ipomoea coccinea and Ipomoea quamoclit, are poor synthesizers with only a few compounds. However, these metabolites are apparently chemotaxonomic markers of this infrageneric taxon in general. They represent either esters of (-)-platynecine (altogether 48 ipangulines and 4 further esters including results of a previous study) or esters of (-)-trachelanthamidine, an additional novel structural type called minalobines (altogether 21 alkaloids). Both types are characterized by section-specific rare necic acids, e.g., ipangulinic/isoipangulinic acid, phenylacetic acid. The alkaloids of Ipomoea cholulensis, I. coccinea, I. hederifolia, Ipomoea neei, and Ipomoea quamoclit were mono and diesters of platynecine. Minalobines turned out to be metabolites of I. lobata (Cerv.) Thell. (syn.: Mina lobata Cerv.) lacking ipangulines. The major alkaloid of this species, minalobine R, has been isolated and identified as 9-O-(threo-2-hydroxy-2-methyl-3-phenylacetoxy-butyryl)-(-)-trachelanthamidine on the basis of spectral data. Apparently only two of the species included in this study, Ipomoea cristulata and Ipomoea sloteri, are able to synthesize both, ipangulines as well as minalobines. Minalobine O could be isolated as a major alkaloid of I. cristulata, its structure has been established as 9-O-(erythro-2-hydroxy-2-methyl-3-tigloyloxy-butyryl)-(-)-trachelanthamidine on the basis of spectral data.     

Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy has been used to structurally characterize the copper-binding site in CusF protein from Escherichia coli. The EPR spectra indicate a single type II copper center with parameters typical for nitrogen and oxygen ligands (A~200 G, g~2.186, g~2.051). The pulsed EPR data show that one of the ligands to Cu²⁺ is an imidazole ring of a histidine residue. The remote amino nitrogen of this imidazole ring is readily observed by electron spin-echo envelope modulation spectroscopy, while the imino nitrogen that is directly coordinated to the Cu²⁺ ion is observed by pulsed electron–nuclear double resonance (ENDOR). In addition, the ENDOR spectra reveal the presence of one more nitrogen ligand that was assigned to be a deprotonated peptide nitrogen. Apart from the two nitrogen ligands, it has been established that there are two nearby hydroxyl protons, although whether these belong to a single equatorial water ligand or two equatorial hydroxide ligands is not known.