Interaction of androsterone and progesterone with inhibitory ligand-gated ion channels: a patch clamp study.

Gamma-aminobutyric acid receptor type A (GABA(A)) receptor channels mediate fast inhibitory neurotransmission throughout the central nervous system while the expression of ionotropic glycine receptors is mainly restricted to the spinal cord and brain stem. Neuroactive steroids are well known as positive allosteric modulators of GABA(A) receptor function. Furthermore, there have been hints for an interaction of neuroactive steroids with ionotropic glycine receptors. The aim of the study was to characterize the effect of androsterone and progesterone on alpha(1) and alpha(1)beta glycine receptor and alpha(1)beta(2)gamma(2) GABA(A) receptor channels and to examine the molecular interactions between ligands and receptors. Electrophysiological recordings were performed on HEK 293 cells using the patch clamp technique in combination with an ultrafast perfusion system. A direct activation of inhibitory ionotropic receptors was observed for androsterone at GABA(A) receptor channels. A coactivation of currents elicited by nonsaturating agonist concentrations was observed with androsterone and progesterone at glycine and GABA(A) receptor channels. We could show that association of beta subunits with alpha subunits affects the sensitivity of glycine receptors to androsterone. In contrast to previous reports in which recombinant glycine receptors were inhibited by progesterone, a potentiating effect was revealed by our experiments. At concentrations of 0.1 mM and higher, there were also hints to a channel block-like mechanism. In conclusion, different molecular mechanisms of interaction between neuroactive steroids and GABA as well as glycine receptors could be identified and quantitatively described. Our data clarify the role of steroid compounds in the modulation of inhibitory receptor channel function.

Functional diversity of recombinant human AMPA type glutamate receptors: possible implications for selective vulnerability of motor neurons.

Lower motor neurons are known to be susceptible to glutamate-mediated cell damage via overstimulation of AMPA type glutamate receptors (GluR). The molecular basis of an important hypothesis in investigating amyotrophic lateral sclerosis (ALS) is glutamate-excitotoxicity. The aim of this study was to define desensitization and deactivation kinetics of recombinant human GluR1 and GluR2 receptor channels and their splice variants by means of patch-clamp experiments employing ultrafast solution exchange techniques. By this approach, the desensitization time constants of homooligomeric channels could be measured as tau(Des)=2.95+/-0.22 ms (n=10) for GluR1flip, tau(Des)=3.17+/-0.19 ms (n=10) for GluR1flop, tau(Des)=9.86+/-0.79 ms (n=10) for GluR2flip, and tau(Des)=1.87+/-0.26 ms (n=10) for GluR2flop, respectively. In the case of GluR1flip/flop and GluR2flop, a nondesensitising steady state current of less than 1% of peak current amplitude was observed, while GluR2flip channel currents showed a marked steady state component of about 10% of the maximum current. No significant differences were detected comparing the deactivation time course of GluR1 and GluR2 splice variants. These results suggest that the human GluR subtypes tested comprise no fundamental difference to their rodent analogous. Therefore, we describe a preparation that will be useful for further investigation of motor neuron physiological properties and a methodological approach allowing to study functional recombinant human GluR channels under reliable conditions.

Quantification of alpha 1-fetoprotein mRNA in peripheral blood and bone marrow: a tool for perioperative evaluation of patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Quantification of alpha 1-fetoprotein (AFP) mRNA in the blood using reverse transcriptase polymerase chain reaction (RT-PCR) could be a useful tool in monitoring the dynamics of minimal residual disease in patients with hepatocellular carcinoma (HCC). Since all available assays do not take into account the efficiency of cell separation, RNA extraction and reverse transcription, a competitive RT-PCR assay for quantification of AFP mRNA in relation to the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) was established. PATIENTS AND METHODS: Peripheral blood of 22 patients and bone marrow aspirates of 11 patients with hepatocellular carcinoma was monitored perioperatively. Eighteen patients with other hepatic tumours or non-malignant hepatic diseases and 26 healthy blood donors served as controls. Messenger RNA contents were calculated relative to the content of GAPDH mRNA as an indicator of total cell count. RESULTS: Among HCC patients, 6 of 22 (26%) were positive for AFP mRNA before operation with values ranging from 2 ag/100 fg to 36 ag/100 fg GAPDH mRNA (mean 14). Among bone marrow samples, AFP mRNA was detectable in 5 of 11 (45%) cases, with 4 ag/100 fg to 23 ag/100 fg GAPDH (mean 9). However, AFP mRNA was also detectable in 3 of 18 (17%) control patients and in 2 of 26 (8%) healthy blood donors. Perioperative findings were highly variable. CONCLUSION: AFP mRNA is not a specific marker for circulating malignant hepatocytes. Whether definition of a cut-off level or the use of a multimarker-PCR will provide more useful data remains to be established.

Characterization of direct readout contacts of the Myb DNA-binding domain.

The Myb protein contacts its recognition sequence by means of direct protein-DNA interactions. We used site-directed mutagenesis in order to substitute amino acids crucial for these contacts and probed the mutant proteins for their DNA-binding and transactiving activities. We could show that amino acids involved in direct readout contacts do not contribute equivalently in the recognition process.

Equine CRISP-3: primary structure and expression in the male genital tract.

Although originally described in the male rodent genital tract, cysteine-rich secretory proteins (CRISPs) are expressed in a variety of mammalian tissue and cell types. The proteins of the male genital tract have been observed associated to spermatozoa and are believed to play a role in mammalian fertilization. Here we describe the identification and primary structure of the first equine member of the CRISP family. Equine CRISP-3 is transcribed and expressed in the stallion salivary gland, in the ampulla and the seminal vesicle. It displays all 16 conserved cysteine residues and shows 82% homology to human and 78% to guinea pig CRISP-2 (AA1, TPX 1) and 77% to human CRISP-3. In contrast to other mammalia, in the horse CRISP-3 is synthesized in great amounts in the accessory sexual glands, ampulla and seminal vesicle, thus allowing the isolation of equine CRISP-3 in amounts suitable for biochemical, physiological and structural studies from stallion seminal plasma.

Mapping of protein-protein interactions between c-myb and its coactivator CBP by a new phage display technique.

We have developed a phage display technique for the mapping of protein-protein interaction sites and characterized the interaction between the c-myb proto-oncogene product and its co-activator CBP. Arbitrary DNA segments of the c-myb gene were cloned into a modified phagemid which allowed for expression in all possible reading frames. The mini-library encompassing all functional domains of the protein was propagated as phages and screened with different bait proteins. Alignment of the sequences revealed that the amino acids 317-342 of Myb interact with the CBP protein. Furthermore, an intramolecular interaction of the N-terminal Myb DNA binding domain with the C-terminus (amino acids 541-567) could be detected.

Sequence context influencing cleavage activity of the K130E mutant of the restriction endonuclease EcoRI identified by a site selection assay.

We have generated several EcoRI mutants which exhibit a decreased cleavage rate on one of the five specific cleavage sites in bacteriophage lambda-DNA. To study the influence of the sequence context on the cleavage rate in more detail, we developed a site selection assay. From a complete set of 4096 plasmid substrates, differing in three bases on both sides of a recognition sequence, optimal (best cut) and unfavorable (worst cut) sequences were selected by repeated limited digestion, separation, and in vivo amplification of cleaved and uncleaved plasmids. In order to compare the sequence preferences of the inner arm mutant K130E and the wild type enzyme, the cleavage rates and sequences of individual plasmids from the resulting pools were determined. The inner arm mutant K130E selected pools with clearly defined consensus sequences and a high amount of palindromic sequences. The cleavage rates of the selected sequences are specific for the K130E mutant as is shown by their cleavage with other mutants. In contrast, wild type EcoRI does not lead to a selection in this assay. Pre-steady state kinetics show that preferences for a certain sequence context are a result of differences in the dissociation rates of the wild type enzyme. EcoRI is evolved to efficiently recognize and cleave each nonmethylated DNA invading the cell. Therefore, a fast dissociation after cleavage is not mandatory.

Accuracy of the EcoRV restriction endonuclease: binding and cleavage studies with oligodeoxynucleotide substrates containing degenerate recognition sequences.

In order to investigate the accuracy of the EcoRV restriction endonuclease, we have synthesized a set of double-stranded oligodeoxynucleotides comprising the canonical recognition sequence, the 9 star sequences (i.e., sequences deviating by one base pair from the canonical sequence), and the 18 mismatch sequences (i.e. sequences deviating by one base from the canonical sequence). For each individual single strand of all these 28 substrates we have measured the rate of phosphodiester bond cleavage under normal buffer conditions. Double-strand cleavage of star substrates is at least 5 orders of magnitude slower than cleavage of the canonical substrate. In contrast, most of the mismatch substrates are accepted more readily. In the absence of the essential cofactor Mg2+, EcoRV binds weakly but equally to the canonical and degenerate substrates, (i.e., KDiss is in the micromolar range). However, the inactive catalytic site mutant D90A in the presence of Mg2+ binds the canonical substrate 1-2 orders of magnitude better than degenerate substrates. Therefore, the EcoRV endonuclease needs the essential cofactor Mg2+ to create thermodynamic discrimination between degenerate and canonical sites. But the main discrimination is kinetically controlled and takes place during cleavage. While in the canonical substrate both single strands are cleaved with an equal velocity, in all other substrates one single strand is cleaved faster than the other one, resulting in a dissociation of the enzyme from the DNA between the two cuts. In vivo this may lead to a repair of the erroneous cleavage site by DNA ligases.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed mutagenesis in the catalytic center of the restriction endonuclease EcoRI.

The catalytic center of the restriction endonuclease (ENase) EcoRI is structurally homologous to that of EcoRV, BamHI and PvuII. Each of these ENases contains a short motif of three to four amino acid (aa) residues which are positioned in a similar orientation to the scissile phosphodiester bond. We have mutated these aa (Pro90, Asp91, Glu111 and Lys113) in EcoRI to determine their individual roles in catalysis. The replacement of Asp91 and Lys113, respectively, by conservative mutations (Ala91, Asn91, Ala113, Gln113, His113 and Leu113) resulted in a reduction of binding affinity and complete loss of cleavage activity. Only Lys113-->Arg substitution still allows to cleave DNA, albeit with a rate reduced by at least four orders of magnitude. Lys113 seems to stabilize the structure of the wild-type (wt) ENase since all five ENase variants with mutations at this position show a strongly enhanced tendency to aggregate. The Ala and Gln mutants of Glu111 bind the recognition sequence slightly stronger than wt EcoRI and cleave it with a low, but detectable rate. Only the Glu111-->Lys mutant, in which the charge is reversed, shows neither binding nor cleavage activity. Pro90 is not important for catalysis, because the Ala90 mutant cleaves DNA with an only slightly reduced rate. Under star conditions, however, this mutant is even more active than wt EcoRI. Therefore, the charged aa Asp91, Glu111 and Lys113 are essential for catalytic activity of the EcoRI ENase.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.

Substrate-assisted catalysis was suggested to be involved in the DNA cleavage reaction of the restriction endonucleases (ENases) EcoRI and EcoRV, because experimental evidence exists that the phosphate group 3' to the scissile bond serves to deprotonate the attacking water. Here, we have addressed the question whether this is a general mechanistic feature of the reactions catalyzed by ENases. For this purpose, the cleavage rates of modified and unmodified oligodeoxyribonucleotides (oligos), in which the phosphate group 3' to the scissile bond is substituted by a methyl phosphonate, were measured for 17 enzymes. Only five turned out not to be inhibited by this modification (BglII, BstI, BstYI, Cfr10I and MunI); all others cleave the modified substrate at a strongly reduced rate or not at all. By employing a hemisubstituted oligo substrate we were able to further investigate the mechanism of inhibition of the latter group of ENases. Some of them cleave the unmodified strand of the modified substrate with a nearly unaltered rate, whereas the modified strand is cleaved very slowly or not at all (BamHI, Bsp143I, Eco72I, MflI, NdeII, Sau3AI, XhoII). The others (AluI, Cfr9I, DpnII, MboI, PvuII) cleave the modified strand of the modified substrate with a largely reduced rate or not at all. These ENases, however, cleave the unmodified strand with a reduced rate, too. Based on these results we conclude that BamHI, Bsp143I, Cfr9I, DpnII, Eco72I, MboI, MflI, NdeII, PvuII, Sau3AI and XhoII may possibly employ substrate assistance in catalysis.

Biophysical characterization of the c-Myb DNA-binding domain.

We have examined proteins containing the DNA-binding domain of c-Myb with biophysical methods. This DNA-binding domain consists of three imperfect repeats (R1, R2, and R3) conserved among many species. Our results indicate that the DNA-binding domain forms unspecific and specific complexes with oligodeoxynucleotides. In the presence of R1, DNA sequences related to a canonical c-Myb-binding site are better discriminated. Furthermore, although R2 and R3 are sufficient for sequence-specific DNA binding, a structural change of the DNA-binding domain upon specific complex formation is induced only when R1 is present. Therefore, R1 might serve as an important element required for secondary structure alteration upon binding and its stabilization as well as for better discrimination between specific and related DNA sequences.

Protein engineering of the restriction endonuclease EcoRV: replacement of an amino acid residue in the DNA binding site leads to an altered selectivity towards unmodified and modified substrates.

According to the crystal structure analysis of a specific EcoRV/DNA complex, the thymine residues of the recognition sequence -GATATC- are not in direct contact with any amino acid residue of the protein. However, several amino acid residues are sufficiently close that it seemed worthwhile trying to create variants of EcoRV with altered specificity by site-directed mutagenesis. Guided by molecular modelling we have replaced. Asn-188 in the catalytic center of EcoRV by Gln to produce a mutant with a relative preference (compared to wild type EcoRV) for substrates in which one thymine of the recognition sequence is replaced by uracil. We have purified and characterized the resulting N188Q mutant. The selectivity value for the engineered enzyme (the ratio of the kcat/KM values for -GATAUC- versus -GATATC-) differs from that of the wild type enzyme by a factor of more than 200.

Pausing of the restriction endonuclease EcoRI during linear diffusion on DNA.

Linear diffusion is a mechanism to accelerate association rates beyond their three-dimensional diffusional limit. It is employed by the restriction endonuclease EcoRI as well as many other proteins interacting with specific DNA sequences to locate their target sites on the macromolecular substrate. In order to investigate biochemical and biophysical details of the linear diffusion process, we have developed a competitive cleavage assay which allows us to assess with great accuracy the influence of sequence, sequence context, and other structural features on the linear diffusion of EcoRI on DNA. We show here that linear diffusion is not a hopping but a sliding movement in which EcoRI follows the helical pitch of the DNA, because it does not "overlook" any cleavage site. Linear diffusion is slowed when EcoRI encounters sites on the DNA which resemble its recognition site ("star" sites). Pauses of up to 20 s are induced, depending on sequence and orientation of the star site. These data suggest that EcoRI can bind to DNA in two binding modes: one tight, specific, and immobile, leading to DNA cleavage, and another one loose and nonspecific, allowing for linear diffusion. Depending on the similarity between the recognition sequence and the DNA sequence being encountered by EcoRI, there will be a continuous transition between these binding modes. Other proteins bound to the DNA and irregular DNA structures such as bent DNA or a triple helix constitute a barrier that cannot easily be passed by EcoRI.

Does a synthetic peptide containing the leucine-zipper domain of c-myb form an alpha-helical structure in solution?

We have examined a synthetic peptide containing the putative leucine zipper domain of the chicken c-myb proto-oncogene using circular dichroism (CD) spectroscopy. The peptide adopts an alpha-helical structure only at low temperatures and in the presence 2,2,2-trifluoroethanol.

Quantitative polymerase chain reaction with oligodeoxynucleotide ligation assay/enzyme-linked immunosorbent assay detection.

Quantitation of nucleic acids by the polymerase chain reaction (PCR) requires coamplification of a control nucleic acid, usually a variant of the sequence to be analyzed, which is added to the sample DNA and amplified in competition. Following PCR, amplified sample and control DNAs are separated by electrophoresis and quantitated, for example by measuring the radioactivity incorporated into the products during the PCR. The need for both electrophoretic separation and radioactive labeling has considerably impeded the use of PCR for routine purposes, e.g., in the clinical laboratory, which requires automatic processing of many samples in parallel. We describe here a quantitative PCR procedure which circumvents electrophoretic separation and detection by radioactivity. It uses a point mutated version of the sample DNA as an internal control. After competitive PCR, amplified sample and control DNA are distinguished by an oligodeoxynucleotide ligation assay (OLA) (Landegren et al., Science 241, 1077-1080, 1988) using two oligodeoxynucleotides, one carrying a biotin-group at the 5'-end and another one with either a digoxigenin (specific for sample DNA) or fluorescein moiety (specific for control DNA), respectively, incorporated close to the 3'-end. Biotinylated oligodeoxynucleotides, educts as well as products of the ligation reaction, are immobilized on avidin-coated microtiter plates. Quantitation of digoxigenin and fluorescein-labeled oligodeoxynucleotide ligation products is achieved by an enzyme-linked immunosorbent assay. This method is very well suited for fast automated or semiautomated PCR.

A fast and accurate enzyme-linked immunosorbent assay for the determination of the DNA cleavage activity of restriction endonucleases.

We have developed an assay procedure to monitor the cleavage of DNA substrates by restriction endonucleases. This procedure uses DNA substrates that are labeled with biotin on one 5' end and with an antigenic group, e.g., fluorescein or digoxigenin, on the other 5' end. After incubation with the restriction enzyme, the reaction is stopped with EDTA and an aliquot is pipetted into the well of an avidin-coated microtiter plate. This immobilizes the unreacted substrate and the biotinylated cleavage product, whereas the other cleavage product labeled with the antigenic group is subsequently washed off. The unreacted substrate is detected by an enzyme-linked immunosorbent assay with an appropriate enzyme-linked antibody. To test our assay we have measured the steady-state rate constants for cleavage of DNA by EcoRI yielding a kcat of 8.6 min-1 and a Km of 150 nM, which are close to values measured with other assays. The advantage of this assay is that it is not only fast and accurate, but also very sensitive. It allows for many samples to be analyzed in parallel and lends itself to automation. Furthermore, this assay can be designed as a competitive assay, when two substrates carrying different antigenic groups are used. The usefulness of such competitive assay is demonstrated by determining the influence of sequence context on the rate of DNA cleavage by EcoRI.

Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes.

The crystal structure analyses of the EcoRI-DNA and EcoRV-DNA complexes do not provide clear suggestions as to which amino acid residues are responsible for the activation of water to carry out the DNA cleavage. Based on molecular modeling, we have proposed recently that the attacking water molecule is activated by the negatively charged pro-Rp phosphoryl oxygen of the phosphate group 3' to the scissile phosphodiester bond. We now present experimental evidence to support this proposal. (i) Oligodeoxynucleotide substrates lacking this phosphate group in one strand are cleaved only in the other strand. (ii) Oligodeoxynucleotide substrates carrying an H-phosphonate substitution at this position in both strands and, therefore, lacking a negatively charged oxygen at this position are cleaved at least four orders of magnitude more slowly than the unmodified substrate. These results are supported by other modification studies: oligodeoxynucleotide substrates with a phosphorothioate substitution at this position in both strands are cleaved only if the negatively charged sulfur is in the RP configuration as shown for EcoRI [Koziolkiewicz, M. & Stec, W.J. (1992) Biochemistry 31, 9460-9466] and EcoRV (B. A. Connolly, personal communication). As the phosphate residue 3' to the scissile phosphodiester bond is not needed for strong DNA binding by both enzymes, these findings strongly suggest that this phosphate group plays an active role during catalysis. This proposal, furthermore, gives a straightforward explanation of why in the EcoRI-DNA and EcoRV-DNA complexes the DNA is distorted differently, but in each case the 3' phosphate group closely approaches the phosphate group that is attacked. Finally, an alternative mechanism for DNA cleavage involving two metal ions is unlikely in the light of our finding that both EcoRI and EcoRV need only one Mg2+ per active site for cleavage.

Determination of the DNA bend angle induced by the restriction endonuclease EcoRV in the presence of Mg2+.

We have used the method of Zinkel and Crothers (Zinkel, S.S., and Crothers, D.M. (1990) Biopolymers 29, 29-38) to determine the degree of bending induced by the binding of the restriction endonuclease EcoRV to its recognition sequence (-GATATC-). A set of four calibration DNA fragments was constructed that contained zero, two, four, or six phased A-tracts in their centers and an EcoRV site at the 5'-end to account for the electrophoretic influence of the bound protein. The mobilities of these calibration molecules complexed with EcoRV were compared to that of a test DNA containing a central EcoRV site also complexed with EcoRV. The EcoRV-induced bend angle was found to be 44 degrees +/- 4 degrees. These experiments were performed with a catalytically inactive EcoRV mutant that still binds DNA specifically in the presence of Mg2+. In the absence of Mg2+, which is necessary for specific binding, there is no difference in the mobilities of the fragments with a peripheral or a central EcoRV site complexed with EcoRV, indicating that nonspecific binding on average does not lead to measurable DNA bending.

Mutational analysis of the function of Gln115 in the EcoRI restriction endonuclease, a critical amino acid for recognition of the inner thymidine residue in the sequence -GAATTC- and for coupling specific DNA binding to catalysis.

The Gln115 residue of the EcoRI restriction endonuclease has been proposed to form a hydrophobic contact to the methyl group of the inner thymidine of the EcoRI recognition sequence -GAATTC- and to be involved in intramolecular hydrogen bonds to the mainchain at positions 140 and 143 as well as to the side-chain of Asn173. We have exchanged Gln115 for Ala and Glu by site-directed mutagenesis and analysed the purified mutant proteins (Q115A and Q115E) biochemically and physico-chemically. Q115A and Q115E have the same secondary structure composition as wild-type EcoRI but are less stable towards thermal denaturation than the wild-type enzyme. In contrast to wild-type EcoRI the mutant proteins show a biphasic denaturation profile under alkaline pH, presumably because the amino acid exchange labilizes one part of the molecule, which unfolds before the rest of the protein is denatured. Q115A is catalytically inactive under normal buffer conditions, in part due to a diminished affinity towards DNA. At low ionic strength and alkaline pH, as well as in the presence of Mn2+, i.e. under conditions where wild-type EcoRI shows a relaxed specificity, Q115A is active, however not as much as wild-type EcoRI. Under these conditions it cleaves the canonical sequence -GAATTC- with the same kcat/Km value as the sequence -GAAUTC-, which differs from the former sequence by a single methyl group, while wild-type EcoRI shows a tenfold lower kcat/Km for cleavage of -GAAUTC- than for -GAATTC-. Binding experiments, carried out in the absence of Mg2+, demonstrate that Q115A has a similar affinity towards -GAATTC- as to -GAAUTC-, while wild-type EcoRI binds to -GAATTC- with a tenfold preference over -GAAUTC-. On the basis of these thermodynamic and kinetic results it can be concluded that the hydrophobic contact between the gamma-methylene group of Gln115 and the methyl group of the inner thymidine contributes about 3 kJ/mol (0.7 kcal/mol) to the energy of interaction, both in the ground and the transition state. Q115E is catalytically inactive under normal buffer conditions, but becomes active at low ionic strength or in the presence of Mn2+. Different from Q115A, Q115E is inactive at alkaline pH and its DNA binding affinity is highest at acidic pH.(ABSTRACT TRUNCATED AT 400 WORDS)