Mitochondrial metabolism of guanine nucleotides. Possible role of guanosine

The catabolism of intramitochondrial guanine nucleotides was examined. During 30 min incubation of rat liver mitochondria at 37 degrees C in the presence of oligomycin and carboxyatractyloside, guanine and xanthine were formed and appeared in the medium. Under these conditions, the direct conversion of GMP to guanine by hypoxanthine-guanine phosphoribosyltransferase is suggested to be the main catabolic route within the organelles. Only very small amounts of guanosine were produced and detected both inside and outside the organelles. [14C]Guanosine and [14C]inosine were taken up by the mitochondria. Therefore, guanosine is suggested to be a precursor of intramitochondrial guanine nucleotides.     

Mutational analysis of conserved nucleotides in a self-splicing group I intron.

We have constructed all single base substitutions in almost all of the highly conserved residues of the Tetrahymena self-splicing intron. Mutation of highly conserved residues almost invariably leads to loss of enzymatic activity. In many cases, activity could be regained by making additional mutations that restored predicted base-pairings; these second site suppressors in general confirm the secondary structure derived from phylogenetic data. At several positions, our suppression data can be most readily explained by assuming non-Watson-Crick base-pairings. In addition to the requirements imposed by the secondary structure, the sequence of the intron is constrained by "negative interactions", the exclusion of particular nucleotide sequences that would form undesirable secondary structures. A comparison of genetic and phylogenetic data suggests sites that may be involved in tertiary structural interactions.     

Comprehensive characterization of molecular interactions based on nanomechanics

Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions.     

Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis

More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3' terminus of the negative strand of the viral genome with a dissociation constant (Kd) of approximately 3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.     

Inter-oligomer interactions of the human prion protein are modulated by the polymorphism at codon 129

The common polymorphism at codon 129 in the human prion protein (PrP) has been shown in many studies to influence not only the pathology of prion disease but also the misfolding propensity of PrP. Here we used NMR, CD and atomic force microscopy in solution to investigate differences in β-oligomer (β^O) formation and inter-oligomer interaction depending on the polymorphism at codon 129. NMR investigations assigned the observable amide resonances to the β^O N-terminal segments, showing that it is the core region of PrP (residues 127-228) that is involved in β^O formation. Atomic force microscopy revealed distinctive 1.8 × 15 × 15-nm disk-like structures that form stacks through inter-oligomer interactions. The propensity to form stacks and the number of oligomers involved depended on the polymorphism at codon 129, with a significantly lower degree of stacking for β^O with valine at position 129. This result provides evidence for conformational differences between the β^O allelic forms, showing that the core region of the protein including position 129 is actively involved in inter-oligomer interactions, consistent with NMR observations.     

Salt processing in larval Drosophila: choice, feeding, and learning shift from appetitive to aversive in a concentration-dependent way

Sodium and chloride need to be ingested and cannot be stored. Therefore, choice of habitat and diet as related to NaCl needs to be tightly regulated. We thus expect that the behavioral effects of salt are organized according to its concentration. Here, we comparatively "fingerprint" the reflex releasing (in choice and feeding experiments) versus the reinforcing effects of sodium chloride ("salt") in terms of their concentration dependencies, using larval Drosophila. Qualitatively, we find that the behavioral effects of salt in all 3 assays are similar: choice, feeding, and reinforcing effect all change from appetitive to aversive as concentration is increased. Quantitatively, however, the appetitive effects for choice and feeding share their optimum at around 0.02 M, whereas the dose-response curve for the reinforcing effect is shifted by more than one order of magnitude toward higher concentrations. Interestingly, a similar shift between these 2 kinds of behavioral effect is also found for sugars (Schipanski et al. 2008). Thus, for salt and for sugar, the sensory-to-motor system is more sensitive regarding immediate, reflexive behavior than regarding reinforcement. We speculate that this may partially be due to a dissociation of the sensory pathways signaling toward either reflexive behavior or internal reinforcement.     

Conformational pH dependence of intermediate states during oligomerization of the human prion protein

Intermediate states are key to understanding the molecular mechanisms governing protein misfolding. The human prion protein (PrP) can follow various misfolding pathways, and forms a soluble beta-sheet-rich oligomer under acidic, mildly denaturing, high salt conditions. Here we describe a fast conformational switch from the native alpha-monomer to monomeric intermediate states under oligomer-forming conditions, followed by a slower oligomerization process. We observe a pH dependence of the secondary structure of these intermediate forms, with almost native-like alpha-helical secondary structure at pH 4.1 and predominantly beta-sheet characteristics at pH 3.6. NMR spectroscopy differentiates these intermediate states from the native protein and indicates dynamic rearrangements of secondary structure elements characteristic of a molten globule. The alpha-helical intermediate formed at pH 4.1 can convert to the beta-sheet conformation at pH 3.6 but not vice versa, and neither state can be reconverted to an alpha-monomer. The presence of methionine rather than valine at codon 129 accelerates the rate of oligomer formation from the intermediate state.     

Hydroxyurea‐induced partial mushroom body ablation does not affect acquisition and retention of olfactory differential conditioning in honeybees

The mushroom bodies (MBs), a paired structure in the insect brain, play a major role in storing and retrieving olfactory memories. We tested whether olfactory learning and odor processing is impaired in honeybees in which MB subunits were partially ablated. Using hydroxyurea (HU) to selectively kill proliferating cells, we created honeybees with varying degrees of MB lesions. Three‐dimensional reconstructions of brains were generated to analyze the drug‐induced morphological changes. These reconstructions show that, with few exceptions, only the MBs were affected by the drug, while other brain areas remained morphometrically intact. Typically, lesions affected only the MB in one hemisphere of the brain. To preclude HU‐induced physiologic deficits in the antennal lobe (AL) affecting olfactory learning, we measured the responses to odors in the AL using an in vivo calcium imaging approach. The response patterns did not differ between the AL of intact versus ablated brain sides within respective specimens. We, therefore, carried out side‐specific classical discriminative olfactory conditioning of the proboscis extension reflex (PER) with control bees and with HU‐treated bees with or without MB ablations. All experimental groups learned equally to discriminate and respond to a rewarded (CS+) but not to an unrewarded (CS?) conditioned stimulus during acquisition and retention tests. Thus, our results indicate that partial MB lesions do not affect this form of elemental olfactory learning. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 343–360, 2002