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• The method of Gadolinium labeled polymeric nanocarriers was developed. • The empty nanocapsules did not affect the viability of the tested cells. • Encapsulated paclitaxel retained its strong cytotoxic/cytostatic activity. • Proposed nanocarriers can serve as an effective contrast agent that can be observed/monitored by MRI. Here, we designed a novel Gadolinium (Gd) labeled drug-loaded polyelectrolyte nanocarriers for theranostics. The nanocarriers were formed via layer-by-layer technique with biodegradable polyelectrolytes: PLL (Poly-L-lysine), PLL-Gd (Gadolinium-labeled Poly-L-lysine) and PGA (Poly-L-glutamic acid). Anticancer drug (Paclitaxel) was encapsulated in the formed nanocarriers. The average size of synthesized nanocarriers was around 150 nm. The empty gadolinium labeled nanocarriers did not show any deleterious effects on tested cells (CT26-CEA, B16F10, 4T1 and PBMC), whereas encapsulated paclitaxel retained its cytotoxic/cytostatic activity. Using T 2 and T 1 NMR relaxation measurements with 9.4 T preclinical MRI scanner, we demonstrated that gadolinium labeled nanocarriers can be detected due to a locally altered contrast in the MR image. Thus, they may become a promising platform for future theranostic applications. [ABSTRACT FROM AUTHOR]

The rational design of novel self-assembled nanomaterials based on peptides remains a great challenge in modern chemistry. A hierarchical approach for the construction of nanofibrils based on α,β-peptide foldamers is proposed. The incorporation of a helix-promoting trans-(1S,2S)-2-aminocyclopentanecarboxylic acid residue in the outer positions of the model coiled-coil peptide led to its increased conformational stability, which was established consistently by the results of CD, NMR and FT-IR spectroscopy. The designed oligomerization state in the solution of the studied peptides was confirmed using analytical ultracentrifugation. Moreover, the cyclopentane side chain allowed additional interactions between coiled-coil-like structures to direct the self-assembly process towards the formation of well-defined nanofibrils, as observed using AFM and TEM techniques.

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrA Hp ) belongs to the well conserved family of serine proteases. HtrA Hp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrA Hp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed Δ htrA mutant strain, we found that bacteria deprived of HtrA Hp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrA Hp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrA Hp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (T m ) values of above 85°C. Moreover, HtrA Hp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrA Hp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope.

Background: Purine-nucleoside phosphorylase (PNP) is known as a tool for the synthesis of various nucleosides and nucleoside analogues. Mechanism, properties, molecular diversity and inhibitors of PNP, particularly these of pharmacological significance, are briefly characterized., Methods: UV and fluorescence spectroscopy was used for kinetic experiments, and HPLC chromatography for product analyses., Results: Applications of various forms of PNP to synthesis of selected fluorescent nucleosides, particularly ribosides of 1,N6-ethenoadenine and various 8-azapurines (triazolo[4,5-d]pyrimidines) are reviewed. Different specificity of various PNP forms is described towards nucleobase and analogue substrates as well as variable ribosylation sites observed in some reactions, with a possibility to further modify these features via the site-directed mutagenesis., Conclusion: Present and future applications of the fluorescent or fluorogenic ribosides are discussed, with particular emphasis on biochemical and clinical analyses with improved sensitivity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Purine and pyrimidine nucleoside phosphorylases catalyze the reversible phosphorolytic cleavage of the glycosidic bond of purine and pyrimidine nucleosides, and are key enzymes of the nucleoside salvage pathway. This metabolic route is the less costly alternative to the de novo synthesis of nucleosides and nucleotides, supplying cells with these important building blocks. Interest in nucleoside phosphorylases is not only due to their important role in metabolism of nucleosides and nucleotides, but also due to the potential medical use of the enzymes (all phosphorylases in activating prodrugs - nucleoside and nucleic base analogs, high-molecular mass purine nucleoside phosphorylases in gene therapy of some solid tumors) and their inhibitors (as selective immunosuppressive, anticancer and antiparasitic agents, and preventing inactivation of other nucleoside drugs). Phosphorylases are also convenient tools for efficient enzymatic synthesis of otherwise inaccessible nucleoside analogues. In this paper the contribution of Professor David Shugar and some of his colleagues and coworkers in studies of these remarkable enzymes carried out over nearly 40 years is discussed on the background of global research in this field.

Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i.e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of GdnHCl. The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of bis-ANS. Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements, that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is formation of molten globule, prone to aggregation.

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with Mr = (90.6 +/- 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02-1.0 mg/ml, presence of up to 100 mM phosphate and up to 200 mM NaCl, temperature in the range 4-25 degrees C. The sedimentation coefficient (6.04 +/- 0.02) S, together with the diffusion coefficient (6.15 +/- 0.11) 10(-7) cm2/s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mM Hepes, pH 7.0, yielded a molecular mass of (90.2 +/- 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 microg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.

The three-dimensional structure of the trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. has been determined by X-ray crystallography. The binary complex of the enzyme with orthophosphate was crystallized in the orthorhombic space group P212121 with unit cell dimensions a=64.1 A, b=108.9 A, c=119.3 A and an enzymatically active trimer in the asymmetric unit. X-ray data were collected at 4 degrees C using synchrotron radiation (EMBL/DESY, Hamburg). The structure was solved by molecular replacement, with the calf spleen PNP structure as a model, and refined at 2.2 A resolution. The ternary "dead-end" complex of the enzyme with orthophosphate and 8-iodoguanine was obtained by soaking crystals of the binary orthophosphate complex with the very weak substrate 8-iodoguanosine. Data were collected at 100 K with CuKalpha radiation, and the three-dimensional structure refined at 2.4 A resolution. Although the sequence of the Cellulomonas PNP shares only 33 % identity with the calf spleen enzyme, and almost no identity with the hexameric Escherichia coli PNP, all three enzymes have many common structural features, viz. the nine-stranded central beta-sheet, the positions of the active centres, and the geometrical arrangement of the ligands in the active centres. Some similarities of the surrounding helices also prevail. In Cellulomonas PNP, each of the three active centres per trimer is occupied by orthophosphate, and by orthophosphate and base, respectively, and small structural differences between monomers A, B and C are observed. This supports cooperativity between subunits (non-identity of binding sites) rather than existence of more than one binding site per monomer, as previously suggested for binding of phosphate by mammalian PNPs. The phosphate binding site is located between two conserved beta- and gamma-turns and consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water molecules. The guanine base is recognized by a zig-zag pattern of possible hydrogen bonds, as follows: guanine N-1...Glu204 O(epsilon1)...guanine NH2...Glu204 O(epsilon2). The exocyclic O6 of the base is bridged via a water molecule to Asn246 N(delta), which accounts for the inhibitory, but lack of substrate, activity of adenosine. An alternative molecular mechanism for catalysis by trimeric PNPs is proposed, in which the key catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) supports binding of 6-oxopurines rather than catalysis. This mechanism, in contrast to that previously suggested, is consistent with the excellent substrate properties of N-7 substituted nucleosides, the specificity of trimeric PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of Glu201/Ala and Asn243/Ala point variants of human PNP., (Copyright 1999 Academic Press.)

A series of 7-alkyl analogues of guanosine was prepared by alkylation of 5'-GMP, and enzymatic dephosphorylation of the products to the corresponding nucleosides. The latter were all excellent, as well as fluorescent, substrates of calf spleen nucleoside phosphorylase. Kinetic parameters demonstrated that the purine ring N(7) is not a binding site for the enzyme.

Under conditions where 2'-deoxycoformycin is enzymatically phosphorylated by wheat shoot phosphotransferase to the 5'-phosphate in 15-20% yield, coformycin is a relatively poor substrate, and is phosphorylated only to the extent of less than or equal to 5%. However, chemical phosphorylation of coformycin by modifications of the Yoshikawa procedure led to isolation of coformycin-5'-phosphate in 20% overall yield. Coformycin-5'-phosphate was characterized by various criteria, including 1H NMR spectroscopy. Comparison of the spectrum with that of the parent nucleoside indicated that the nucleotide is predominantly, although not exclusively, in the conformation anti about the glycosidic bond. Like 2'-deoxycoformycin-5'-phosphate, coformycin-5'-phosphate was a feeble substrate of snake venom 5'-nucleotidase, and is hydrolyzed, quantitatively, at only 2% the rate for 5'-AMP. With 5'-AMP analogues as substrate, the 5'-phosphates of both coformycin and deoxycoformycin were poor inhibitors of the enzyme, with Ki values greater than 0.3 mM. The 5'-phosphates of both coformycin and deoxycoformycin do not significantly inhibit adenosine deaminase (Ki greater than 0.2 mM), but are potent inhibitors of adenylate deaminase (Ki less than or equal to 10(-9) M). Neither coformycin nor deoxycoformycin are inhibitors of mammalian purine nucleoside phosphorylase. The stabilities of coformycin, deoxycoformycin, and their 5'-phosphates, have been examined as a function of pH, and nature of the buffer medium. In particular, all exhibit instability in acid and neutral media, but are relatively stable in the vicinity of pH 9. Some biological aspects of the overall results are presented.