Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

 A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM^–1 s^–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature ¹⁷O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA. Received: 27 January 1997 / Accepted: 12 May 1997     

Metal-modified nucleobase pairs involving 7,9-dimethylguanine: trans-a2PtII analogues (a = NH3 or CH3NH2) of Watson-Crick GC and homo GG pairs

 The analogy between H-bonded nucleobase pairs and their metalated analogues is extended to the hemiprotonated pair of 7,9-dimethylguanine (7,9-DimeG) and the Watson-Crick and reversed Watson-Crick pair between 7,9-dimethylguaninium (7,9-DimeGH⁺) and 1-methylcytosine (1-MeC). The crystal structure analyses of two model compounds, trans–[Pt(CH₃NH₂)₂(7,9-DimeG-N1)₂](NO₃)₂ (1) and trans–[Pt(NH₃)₂(1-MeC-N3)(7, 9-DimeG-N1)](PF₆)₂· 2.5 H₂O (3a) are reported. Pt binding is through N1 of 7,9-DimeG and N3 of 1-MeC. In solution, 3a exists in a mixture with Watson-Crick and reversed Watson-Crick arrangements of the two bases, depending on solvent, concentration and anions. Received: 16 October 1996 / Accepted: 27 January 1997     

Novel findings on the copper catalysed oxidation of cysteine

Summary The oxidation of cysteine (RSH) has been studied by using O₂, ferricytochrome c (Cyt c) and nitro blue tetrazolium (NBT) as electron acceptors. The addition of 200M Cu^II to a solution of 2mM cysteine, pH 7.4, produces an absorbance with a peak at 260 nm and a shoulder at 300 nm. Generation of a cuprous bis-cysteine complex (RS-Cu^I-SR) is responsible for this absorbance. In the absence of O₂ the absorbance is stable for long time while in the presence of air it vanishes slowly only when the cysteine excess is consumed. The neocuproine assay and the EPR analysis show that the metal remains reduced in the course of the oxidation of cysteine returning to the oxidised form at the end of reaction when all RSH has been oxidised to RSSR. Addition of Cu^II enhances the reduction rate of Cyt c and of NBT by cysteine also under anaerobiosis indicating the occurrence of a direct reduction of the acceptor by the complex. It is concluded that the cuprous bis-cysteine complex (RS-Cu^I-SR) is the catalytic species involved in the oxidation of cysteine. The novel finding of the stability of the complex together with the metal remaining in the reduced form during the oxidation suggest sulfur as the electron donor in the place of the metal ion.Abbreviations RSH cysteine - RS^– cysteine in the thiolate form - RS^· thiyl radical of cysteine - RSSR cystine - Cyt c cytochrome c - SOD superoxide dismutase - NBT nitro blue tetrazolium - NBF nitro blue formazan - DTNB 5,5-dithiobis-2-nitrobenzoic acid - DTPA diethylenetriaminepentaacetic acid Dedicated to prof. A. Ballio ob the occasion of his 75th birthday.     

Roles of ubiquitin-mediated proteolysis in cell cycle control

Selective degradation of cyclins, inhibitors of cyclin-dependent kinases and anaphase inhibitors is responsible for several major cell cycle transitions. The degradation of these cell cycle regulators is controlled by the action of ubiquitin—protein-ligase complexes, which target the regulators for degradation by the 26S proteasome. Recent results indicate that two types of multisubunit ubiquitin ligase complexes, which are connected to the protein kinase regulatory network of the cell cycle in different ways, are responsible for the specific and programmed degradation of many cell cycle regulators.     

On the RNA World: Evidence in Favor of an Early Ribonucleopeptide World

A highly complex RNA world, as is sometimes presented in view of the widespread and diversified use of RNA enzymes, would have encountered many difficulties in passing to a world with catalysis mediated by proteins. These difficulties can be overcome by postulating a very early relationship between the nucleotide and the amino acid components. In particular, after asserting that some characteristics expressed by (nucleotide) coenzymes in catalysis are easier to understand if a close and early relationship between these coenzymes and amino acids is hypothesized, a model is presented for the origin of the enzyme–coenzyme complex. This model is essentially based on an intermediate formed by a tRNA-like molecule covalently linked to a polypeptide. The model attributes the majority of the catalytic role in the ribonucleoprotein world to the latter complex and thus it takes into account the birth of the key intermediate in the origin of protein synthesis—namely, peptidyl-tRNA, which would have otherwise been extremely difficult to select. The predictions of the model are discussed along with its robustness, using the data derived from the study of intermediary metabolism and those from molecular biology. Finally, the appearance of the genetic code in the late phase of the ribonucleopeptide world is discussed. Received: 13 January 1997 / Accepted: 25 July 1997     

Resolution of sensory and mucoid glycoconjugates with terminal α-galactose residues in the mucomicrovillar complex of the vomeronasal sensory epithelium by dual confocal laser scanning microscopy

The organization of the mucomicrovillar complex of the vomeronasal sensory epithelium of adult rats was examined using confocal laser scanning microscopy. In specimens labeled with the FITC-conjugated isolectin B₄ of Bandeiraea simplicifolia, which recognizes terminal -galactose sugar residues of glycoconjugates, we demonstrated that the mucomicrovillar complex was composed of islet-like structures with a high-density -galactose core. The mucomicrovillar complex was further resolved into sensory and mucoid components in double-labeling and dual scanning experiments. The sensory component, which consists of the dendritic terminals of olfactory marker protein-immunoreactive vomeronasal receptor neurons, contained cytosolic glycoconjugates with terminal -galactose sugar residues. The extracellular mucoid component consisted of glycoconjugates containing terminal -galactose derived from the glands associated with the vomeronasal organ. These results demonstrated the complex microchemical organization of the sensory and mucoid components of the mucomicrovillar complex.     

Functional organization of the macroglomerular complex related to behaviourally expressed olfactory redundancy in male cabbage looper moths

Abstract. The neurophysiological bases for behaviourally expressed olfactory redundancy in the sex pheromone communication system of the cabbage looper moth, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), were examined by coupling the cut-sensillum extracellular recording technique with a highly specific neuronal marking method for moth peripheral receptors. In seventy-two antennal sensilla, axonal pathways of cobalt-stained neurones could be traced into the male-specific macroglomerular complex in the antennal lobe. In T. ni males this comprises five glomeruli, two of which are subdivided into morphologically, and in some instances functionally identifiable, regions. Axonal arborizations of forty-eight neurones (single stainings) showed high fidelity (98%) for containment within a specific glomerulus or glomerular subdivision, and the neuropil targeted seemed to be related to the specificity of a neurone to a particular female-emitted sex pheromone component (27-12:Ac, Z7-14:Ac, Z9-14:Ac, 12:Ac, 11–12:Ac, Z5-12:Ac), or to a behavioural antagonist (Z7-12:OH). Double (twenty-one) and multiple stainings (three) showed axons projecting into two or more glomeruli, respectively, with 100% fidelity for the component-specific glomerulus or glomerular subdivision to be targeted. We suggest that the potential for a single minor component to cross-stimulate two or more neurones within a sensillum may enable partial blends to continue to provide sensory input into all of the pheromone-processing glomeruli of the complex. Our interpretation is that redundancy occurs at the receptor level on neighbouring dendrites, and thus allows various four-component partial blends to evoke full pheromone-mediated behaviour.     

Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.