Synthesis,conformational study,and spectroscopic characterization of the cyclic Cα,α‐disubstituted glycine 9‐amino‐9‐fluorenecarboxylic acid

A series of terminally blocked peptides (to the pentamer level) from l ‐Ala and the cyclic C^α,α‐disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc‐Afc‐OMe and the dipeptide Boc‐Afc‐Gly‐OMe were determined in the crystal state by X‐ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT‐IR absorption and ¹H‐NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully‐extended (C₅) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C^α,α‐disubstituted Gly residues Ac₅c and Dϕg is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C^α,α‐disubstituted Gly residue is also reported. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Extracellular,low-affinity β-N-acetylglucosaminidases linked to the dynamics of diatoms and crustaceans in freshwater systems of different trophic degree

Extracellular hydrolysis of 4-methylumbelliferyl β-N-acetylglucosaminide was measured in the oligomesotrophic Piburger See and the eutrophic Římov reservoir during spring and summer phytoplankton blooms, respectively. Total enzymatic activity (TEA) ranged between 0.2 and 19.1 nmol 1⁻¹ h⁻¹ in the reservoir and between 0.8 and 12.4 nmol 1⁻¹ h⁻¹ in the lake. High-affinity (K_m < 1 μmol 1⁻¹) and lowaffinity (K_m > 100 μmol 1⁻¹) enzymes were kinetically identifiable in most samples from both localities. The low-affinity enzyme activity (LEA) usually accounted for >60% (mean: 80%) of TEA. LEA and diatom biomass significantly correlated over time in the reservoir epilimnion (r_s = 0.578) and in the lake metalimnion (r_s = 0.862). As diatoms possess chitin and take up its monomer, N-acetylglucosamine, two explanations of the observed relationships are suggested: extracellular β-N-acetylglucosaminidase activity partly originates either from ectoenzymes of chitinolytic bacteria attached to diatom cells or from ectoenzymes of diatoms, enabling them to take up N-acetylglucosamine from ambient amino sugars instead of synthesizing it de novo. A significant positive correlation of LEA with crustacean abundance was found in the lake epilimnion (r_s = 0.850), apparently reflecting the growing spring populations of frequently moulting juvenile crustaceans. A possible contribution of chitinolytic bacteria, accompanying the crustacean populations, to LEA is discussed.     

Antioxidant and detoxifying enzymes in the liver and kidney of pheasants after intoxication by herbicides MCPA and ANITEN

The activity of antioxidant and detoxifying enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase, glutathione-S-transferase (GST), the contents of thiobarbituric acid reactive substances, and the superoxide dismutase and glutathione-S-transferase isoenzyme patterns, were determined in the liver and kidney of pheasants after acute intoxication by herbicides MCPA and ANITEN I. In the liver, the activity of antioxidant enzymes was significantly decreased in the group given ANITEN I. New superoxide dismutase isoforms (pI 6.30, 6.85, 7.00) and higher intensity of isoform with pI 6.60 were observed after isoelectrofocusing in all experimental groups. In the kidney, the activity of superoxide dismutase was significantly decreased, and a higher intensity of superoxide dismutase isoforms (pI 6.00 and 6.60) was observed in all experimental groups. The contents of thiobarbituric acid reactive substances were significantly increased in the group with ANITEN I. The glutathione-S-transferase isoenzyme pattern was studied by using subunit-specific substrates and by Western blotting. The activity of glutathione-S-transferase with ethacrynic acid and cross-reactivity with rat subunit 7 was lower in all experimental groups in the kidney and liver, except in the liver of the group given a higher dose of ANITEN I. In this group, we have found a 2.10-fold higher activity to ethacrynic acid and a strong induction of subunit 7. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 235–244, 1998     

Systems biology of persistent infection: tuberculosis as a case study

The human immune response does an excellent job of clearing most of the pathogens that we encounter throughout our lives. However, some pathogens persist for the lifetime of the host. Despite many years of research, scientists have yet to determine the basis of persistence of most pathogens, and have therefore struggled to develop reliable prevention and treatment strategies. Systems biology provides a new and integrative tool that will help to achieve these goals. In this article, we use Mycobacterium tuberculosis as an example of how systems-biology approaches have begun to make strides in uncovering important facets of the host-pathogen interaction.     

The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast

Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a wide variety of proteins. Two of the best characterized functions of protein ubiquitination are to mark proteins for degradation by cytosolic proteasome and to promote the internalization of certain plasma membrane proteins via the endocytotic pathway, followed by their degradation in the vacuole. Recent studies of membrane proteins both in yeast and mammalian cells suggest that the role of ubiquitin may extend beyond its function as an internalization signal in that it also may be required for modification of some component(s) of the endocytotic machinery, and for cargo protein sorting at the late endosome and the Golgi apparatus level. In this review, I will attempt to bring together what is currently known about the role of ubiquitination in controlling protein trafficking between the yeast plasma membrane, the trans-Golgi network, and the vacuole/lysosome.     

A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET—a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.Subject terms: Myeloma, Cancer therapeutic resistance     

MK17, a specific marker closely linked to the gynoecium suppression region on the Y chromosome in Silene latifolia

The aim of this work was to isolate new DNA markers linked to the Silene latifolia Y chromosome. To do this we created a chromosome-specific plasmid library after DOP-PCR amplification of laser-microdissected Y-chromosomes. The library screening led to the isolation of several clones yielding mostly to exclusive male specific hybridization signals. Subsequent PCR confirmed the Y-unique linkage for one of the sequences. This DNA sequence called MK17 has no homology to any known DNA sequence and it is not expressed. Based on PCR and Southern analyses, MK17 is present only in dioecious species of the Elisanthe section of the genus Silene (S. latifolia, S. dioica, and S. diclinis) and it is absent in related gynodioecious and hermaphroditic species. The mapping analysis using a panel of deletion mutants showed that MK17 is closely linked to the region controlling suppression of gynoecium development. Hence MK17 represents a valuable marker to isolate genes controlling the gynoecium development suppression on the Y chromosome of S. latifolia.     

Morphological and Compositional Shifts in an Experimental Bacterial Community Influenced by Protists with Contrasting Feeding Modes

In a two-stage continuous-flow system, we studied the impacts of different protozoan feeding modes on the morphology and taxonomic structure of mixed bacterial consortia, which were utilizing organic carbon released by a pure culture of a Rhodomonas sp. grown on inorganic medium in the first stage of the system. Two of three second stages operated in parallel were inoculated by a bacterivorous flagellate, Bodo saltans, and an algivorous ciliate, Urotricha furcata, respectively. The third vessel served as a control. In two experiments, where algal and bacterial populations grew at rates and densities typical for eutrophic waters, we compared community changes of bacteria, algae, and protozoa under quasi-steady-state conditions and during the transient stage after the protozoan inoculation. In situ hybridization with fluorescent oligonucleotide probes and cultivation-based approaches were used to tentatively analyze the bacterial community composition. Initially the cell size distribution and community structure of all cultivation vessels showed similar patterns, with a dominance of 1- to 2.5-(mu)m-long rods from the beta subdivision of the phylum Proteobacteria ((beta)-Proteobacteria). Inoculation with the ciliate increased bacterial growth in this substrate-controlled variant, seemingly via a recycling of nutrients and substrate released by grazing on algae, but without any detectable effect on the composition of bacterial assemblage. In contrast, an inoculation with the bacterivore, B. saltans, resulted in a decreased proportion of the (beta)-Proteobacteria. One part of the assemblage (<4% of total bacterial numbers), moreover, produced large grazing-resistant threadlike cells. As B. saltans ingested only cells of <3 (mu)m, this strategy yielded a refuge for (symbl)70% of total bacterial biomass from being grazed. Another consequence of the heavy predation in this variant was a shift to the numerical dominance of the (alpha)-Proteobacteria. The enhanced physiological status of the heavily grazed-upon segment of bacterial community resulted in a much higher proportion of CFU (mean, 88% of total bacterial counts) than with other variants, where CFU accounted for (symbl)30%. However, significant cultivation-dependent shifts of the bacterial community were observed toward (gamma)-Proteobacteria and members of the Cytophaga/Flavobacterium group, which demonstrated the rather poor agreement between cultivation-based approaches and oligonucleotide probing.