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According to general doctrine canceroselectivity of Cyclophosphamide is based on different activities of the 4-hydroxycyclophosphamide (OHCP) detoxifying cellular enzyme aldehyde dehydrogenase in tumor and normal cells. Aldehyde dehydrogenase converts the OHCP tautomere aldophosphamide (ALDO) to the non-cytotoxic carboxyphosphamide. Due to different activities of the detoxifying enzyme more cytotoxic phosporamide mustard (PAM) is spontaneously released from OHCP/ALDO in tumor cells. PAM unfolds its cytotoxic activity by forming intrastrand and interstrand DNA crosslinks. This hypothesis is supported by in vitro experiments which show inverse correlations of aldehyde dehydrogenase activity and sensitivity of tumor cells against activated congeners of cyclophosphamide like mafosfamide which hydrolyses within a few minutes to OHCP. In protein free rat serum ultrafiltrate however free OHCP and its coexisting tautomer ALDO are stable compounds. Its half-life in protein free rat serum ultrafiltrate (pH7, 37oC) is more than 20 h. Contrary to protein free ultrafiltrate in whole serum ALDO is enzymatically decomposed to PAM and 3-hydroxypropionaldehyde (HPA) within minutes. The decomposing enzyme was identified as 3´-5´ phosphodiesterase, the Michaelis constant was determined to be 10-3 M in human serum.
The experiments presented clearly demonstrate that ALDO is not only cleaved base catalyzed yielding acrolein and PAM but also cleaved enzymatically by serum phosphodiesterases yielding HPA and PAM. It is discussed that the reason of the high canceroselectivity of cyclophosphamide is not only due to enrichment of OHCP/ALDO in tumor cells due to less detoxification of ALDO in tumor cells than in normal cells. It is discussed that there is a good reason for an additional mechanism namely the amplification of apoptosis of PAM damaged cells by HPA.
A two step mechanism for the mechanism of action of OHCP/ALDO is discussed. During the first step, the DNA is damaged by alkylation by PAM. During the second step the cell containing damaged DNA is eliminated by apoptosis, supported by HPA.
Fatty acids (FAs) are considered strategically important platform compounds that can be accessed by sustainable microbial approaches. Here we report the reprogramming of chain-length control of Saccharomyces cerevisiae fatty acid synthase (FAS). Aiming for short-chain FAs (SCFAs) producing baker’s yeast, we perform a highly rational and minimally invasive protein engineering approach that leaves the molecular mechanisms of FASs unchanged. Finally, we identify five mutations that can turn baker’s yeast into a SCFA producing system. Without any further pathway engineering, we achieve yields in extracellular concentrations of SCFAs, mainly hexanoic acid (C6-FA) and octanoic acid (C8-FA), of 464 mg l−1 in total. Furthermore, we succeed in the specific production of C6- or C8-FA in extracellular concentrations of 72 and 245 mg l−1, respectively. The presented technology is applicable far beyond baker’s yeast, and can be plugged into essentially all currently available FA overproducing microorganisms.
The field of dynamic nuclear polarization has undergone tremendous developments and diversification since its inception more than 6 decades ago. In this review we provide an in-depth overview of the relevant topics involved in DNP-enhanced MAS NMR spectroscopy. This includes the theoretical description of DNP mechanisms as well as of the polarization transfer pathways that can lead to a uniform or selective spreading of polarization between nuclear spins. Furthermore, we cover historical and state-of-the art aspects of dedicated instrumentation, polarizing agents, and optimization techniques for efficient MAS DNP. Finally, we present an extensive overview on applications in the fields of structural biology and materials science, which underlines that MAS DNP has moved far beyond the proof-of-concept stage and has become an important tool for research in these fields.
The transcriptional regulator far upstream binding protein 1 (FUBP1) is essential for fetal and adult hematopoietic stem cell (HSC) self-renewal, and the constitutive absence of FUBP1 activity during early development leads to embryonic lethality in homozygous mutant mice. To investigate the role of FUBP1 in murine embryonic stem cells (ESCs) and in particular during differentiation into hematopoietic lineages, we generated Fubp1 knockout (KO) ESC clones using CRISPR/Cas9 technology. Although FUBP1 is expressed in undifferentiated ESCs and during spontaneous differentiation following aggregation into embryoid bodies (EBs), absence of FUBP1 did not affect ESC maintenance. Interestingly, we observed a delayed differentiation of FUBP1-deficient ESCs into the mesoderm germ layer, as indicated by impaired expression of several mesoderm markers including Brachyury at an early time point of ESC differentiation upon aggregation to EBs. Coculture experiments with OP9 cells in the presence of erythropoietin revealed a diminished differentiation capacity of Fubp1 KO ESCs into the erythroid lineage. Our data showed that FUBP1 is important for the onset of mesoderm differentiation and maturation of hematopoietic progenitor cells into the erythroid lineage, a finding that is supported by the phenotype of FUBP1-deficient mice.
We have determined the crystal structures of two decachlorocyclopentasilanes, namely bis(tetra-n-butylammonium) dichloride decachlorocyclopentasilane dichloromethane disolvate, 2C16H36N+·2Cl−·Si5Cl10·2CH2Cl2, (I), and bis(tetraethylammonium) dichloride decachlorocyclopentasilane dichloromethane disolvate, 2C8H20N+·2Cl−·Si5Cl10·2CH2Cl2, (II), both of which crystallize with discrete cations, anions, and solvent molecules. In (I), the complete decachlorocyclopentasilane ring is generated by a crystallographic twofold rotation axis. In (II), one cation is located on a general position and the other two are disordered about centres of inversion. These are the first structures featuring the structural motif of a five-membered cyclopentasilane ring coordinated from both sides by a chloride ion. The extended structures of (I) and (II) feature numerous C—H⋯Cl interactions. In (II), the N atoms are located on centres of inversion and as a result, the ethylene chains are disordered over equally occupied orientations.
The asymmetric unit of the title compound, C18H18I2N2O2, consists of one half-molecule, completed by the application of inversion symmetry. The molecule adopts the typical structure for this class of bis-benxozazines, characterized by an anti orientation of the two benzoxazine rings around the central C—C bond. The oxazinic ring adopts a half-chair conformation. In the crystal, molecules are linked by C—I⋯N short contacts [I⋯N = 3.378 (2) Å], generating layers lying parallel to the bc plane.
Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes.
The asymmetric unit of the title co-crystalline adduct, 1,3,6,8-tetraazatricyclo[4.4.1.13,8]dodecane (TATD)–4-iodophenol (1/2), C8H16N4·2C6H5IO, comprises a half molecule of the aminal cage polyamine plus a 4-iodophenol molecule. A twofold rotation axis generates the other half of the adduct. The components are linked by two intermolecular O—H⋯N hydrogen bonds. The adducts are further linked into a three-dimensional framework structure by a combination of N⋯I halogen bonds and weak non-conventional C—H⋯O and C—H⋯I hydrogen bonds.
In the title compound, C26H24N2O2, the oxazine moiety is fused to a naphthalene ring system. The asymmetric unit consists of one half of the molecule, which lies about an inversion centre. The C atoms of the ethylene spacer group adopt an antiperiplanar arrangement. The oxazine ring adopts a half-chair conformation. In the crystal, supramolecular chains running along the b axis are formed via short C—H⋯π contacts. The crystal studied was a non-merohedral twin with a fractional contribution of 0.168 (2) of the minor twin component.
In the title compound, C17H18N2O, the central carbon atom with the OH substituent and one of the (E)-benzylideneamino substituents are disordered over two sets of sites with occupancies of 0.851 (4) and 0.149 (4). The relative positions of the two disorder components is equivalent to a rotation of approximately 60° about the C—N single bond. In the crystal, the molecules are held together by O—H...N hydrogen bonds, forming simple C(5) chains along the b-axis direction. In addition, pairs of the chains are further aggregated by weak C—H...π interactions.
CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin
(2017)
Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane.
Background: How a dentist works, such as the patterns of movements performed daily, is also largely affected by the workstation Dental tasks are often executed in awkward body positions, thereby causing a very high degree of strain on the corresponding muscles. The objective of this study is to detect those dental tasks, during which awkward postures occur most frequently. The isolated analysis of static postures will examine the duration for which these postures are maintained during the corresponding dental, respectively non-dental, activities.
Methods: 21 (11f/10 m) dentists (age: 40.1 ± 10.4 years) participated in this study. An average dental workday was collected for every subject. To collect kinematic data of all activities, the CUELA system was used. Parallel to the kinematic examination, a detailed computer-based task analysis was conducted. Afterwards, both data sets were synchronized based on the chronological order of the postures assumed in the trunk and the head region. All tasks performed were assigned to the categories "treatment" (I), "office" (II) and "other activities" (III). The angle values of each body region (evaluation parameter) were examined and assessed corresponding to ergonomic standards. Moreover, this study placed a particular focus on static positions, which are held statically for 4 s and longer.
Results: For "treatment" (I), the entire head and trunk area is anteriorly tilted while the back is twisted to the right, in (II) and (III) the back is anteriorly tilted and twisted to the right (non-neutral position). Static positions in (I) last for 4–10s, static postures (approx. 60%) can be observed while in (II) and (III) in the back area static positions for more than 30 s are most common. Moreover, in (II) the back is twisted to the right for more than 60 s in 26.8%.
Conclusion: Awkward positions are a major part of a dentists’ work. This mainly pertains to static positions of the trunk and head in contrast to "office work." These insights facilitate the quantitative description of the dentist profession with regard to the related physical load along with the health hazards to the musculoskeletal system. Moreover, the results allow for a selective extraction of the most unfavorable static body positions that dentists assume for each of the activities performed.
Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.
Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.
N-Allyltetramethylpiperidine is readily isomerized to the corresponding enamine by treatment with catalytic amounts of B(C6F5)3. It adds HB(C6F5)2 at the nucleophilic enamine carbon atom to form a C/B Lewis adduct. This reacts with two molar equivalents of carbon monoxide by selective head to tail coupling to give a five-membered C2O2B heterocycle. In contrast the enamine/HB(C6F5)2 Lewis pair reacts with two molar equiv. of nitric oxide by head to head coupling. This reaction probably proceeds via equilibrium with the corresponding vicinal N/B Lewis pair. Most products were characterized by X-ray diffraction.
Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function).
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7 Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7 Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this ‘worm arrhythmia’ by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.
The asymmetric unit of the title compound, C21H28N4O, consists of two unique molecules linked by an O—H⋯N hydrogen bond. The conformation of both C=N bonds is E and the azomethine functional groups lie close to the plane of their associated benzene rings in each of the independent molecules. The dihedral angles between the two benzene rings are 83.14 (4) and 75.45 (4)°. The plane of the one of the N(CH3)2 units is twisted away from the benzene ring by 18.8 (2)°, indicating loss of conjugation between the lone electron pair and the benzene ring. In the crystal structure, O—H⋯N hydrogen bonds together with C—H⋯O hydrogen bonds link neighbouring supramolecular dimers into a three-dimensional network.