HERC3 binding to and regulation by ubiquitin

Members of the HERC (domain homologous to E6 associated protein carboxy-terminus and RCC1 domain protein) family may function both as guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we identify an unstudied member, HERC3. This protein was recognized by specific antibodies in different cell types. HERC3 was located in the cytosol and in vesicular-like structures containing beta-COP, ARF and Rab5 proteins. Involvement of HERC3 in the ubiquitin system was suggested by its ability to interact with ubiquitin. The conserved cysteine in HECT proteins was not essential for this non-covalent binding. Moreover, HERC3 was a substrate of ubiquitination being degraded by the proteasome. These observations indicate a fine regulation of HERC3 and suggest a role in vesicular traffic and ubiquitin-dependent processes.     

The chemokine interleukin-8 acutely reduces Ca(2+) currents in identified cholinergic septal neurons expressing CXCR1 and CXCR2 receptor mRNAs

The chemokine IL-8 is known to be synthesized by glial cells in the brain. It has traditionally been shown to have an important role in neuroinflammation but recent evidence indicates that it may also be involved in rapid signaling in neurons. We investigated how IL-8 participates in rapid neuronal signaling by using a combination of whole-cell recording and single-cell RT-PCR on dissociated rat septal neurons. We show that IL-8 can acutely reduce Ca(2+) currents in septal neurons, an effect that was concentration-dependent, involved the closure of L- and N-type Ca(2+) channels, and the activation of G(ialpha1) and/or G(ialpha2) subtype(s) of G-proteins. Analysis of the mRNAs from the recorded neurons revealed that the latter were all cholinergic in nature. Moreover, we found that all cholinergic neurons that responded to IL-8, expressed mRNAs for either one or both IL-8 receptors CXCR1 and CXCR2. This is the first report of a chemokine that modulates ion channels in neurons via G-proteins, and the first demonstration that mRNAs for CXCR1 are expressed in the brain. Our results suggest that IL-8 release by glial cells in vivo may activate CXCR1 and CXCR2 receptors on cholinergic septal neurons and acutely modulate their excitability by closing calcium channels.     

Fingerprinting of Mixed Bacterial Strains and BIOLOG Gram-Negative (GN) Substrate Communities by Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities. Received: 11 September 1998 / Accepted: 29 October 1998     

Effect of base stacking on the relative thermodynamic stability of oligonucleotide complexes: a spectroscopic study

Three-strand oligonucleotide complexes are employed to assess the effect of base stacking and base pair mismatch on the relative thermodynamic stabilities of oligonucleotide duplexes. The melting behavior of three-strand oligonucleotide complexes incorporating nicks and gaps as well as internal single base mismatches is monitored using temperature-dependent optical absorption spectroscopy. A sequential three-state equilibrium model is used to analyze the measured melting profiles and evaluate thermodynamic parameters associated with dissociation of the complexes. The free-energy of stabilization of a nick complex compared to a gap complex due to base stacking is determined to be -1.9 kcal/mol. The influence of a mispaired base in these systems is shown to destabilize a nick complex by 3.1 kcal/mol and a gap complex by 2.8 kcal/mol, respectively.     

Comparison of two alternative dominant selectable markers for wine yeast transformation

Genetic improvement of industrial yeast strains is restricted by the availability of selectable transformation markers. Antibiotic resistance markers have to be avoided for public health reasons, while auxotrophy markers are generally not useful for wine yeast strain transformation because most industrial Saccharomyces cerevisiae strains are prototrophic. For this work, we performed a comparative study of the usefulness of two alternative dominant selectable markers in both episomic and centromeric plasmids. Even though the selection for sulfite resistance conferred by FZF1-4 resulted in a larger number of transformants for a laboratory strain, the p-fluoro-DL-phenylalanine resistance conferred by ARO4-OFP resulted in a more suitable selection marker for all industrial strains tested. Both episomic and centromeric constructions carrying this marker resulted in transformation frequencies close to or above 10(3) transformants per microg of DNA for the three wine yeast strains tested.     

Synthesis and properties of oligonucleotides carrying cryptolepine derivatives

A carboxy derivative of the antimalarial cytotoxic drug cryptolepine was introduced into synthetic oligonucleotides by reaction of the carboxy derivative of cryptolepine with oligonucleotides carrying an amino group either at the 3'- or at the 5'-end. Oligonucleotides carrying the cryptolepine derivative bind their complementary sequences with greater affinity than unmodified ones. When cryptolepine is attached to a polypyrimidine oligonucleotide designed to form a parallel triplex, the triplex shows none or weak stabilization.     

Synthesis of oligoribonucleotides containing 4-thiouridine using the convertible nucleoside approach and the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group

Oligoribonucleotides containing 4-thiouridine were prepared using the Fpmp group for protection of the 2'-OH. Two uridine derivatives with the 1,2,4-triazolyl and the 2-nitrophenyl groups at position 4 were used to obtain 4-thiouridine by postsynthetic substitution with sodium hydrogen sulfide. Both uridine derivatives allow the preparation of the desired oligonucleotides in good yields.