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PubMed1979

Diffusion of ions in myelinated nerve fibers.

The diffusion of ions towards or away from the inner side of the nodal membrane in preparations, the cut ends of which are placed in various media, was investigated. The ion concentration changes were calculated by numerical solution of the unidimensional electrodiffusion equation under a variety of media compositions, axoplasmic diffusion coefficients, and internal anionic compositions. The potassium and cesium ion diffusion along the axon towards the node was determined experimentally by two different electrophysiological methods. On the basis of comparison between the experimental data and the computational predictions the axoplasmic potassium ion diffusion coefficient was determined to be almost equal to that in free aqueous solution, while that of cesium ion was close to one half of that in aqueous solution. Utilizing the values of diffusion parameters thus determined, we solved the electrodiffusion equation for a number of common experimental procedures. We found that in short fibers, cut 0.1-0.2 cm at each side of the node, the concentration approached values close to the new steady-state values within 5-30 min. In long fibers (over 1 cm long) steady-state concentrations were obtained only after a few hours. Under some conditions the internal concentrations transiently overshot the steady-state values. The diffusion potentials generated in the system were also evaluated. The ion concentration changes and generation of diffusion potential cannot be prevented by using side pools with cation content identical to that of the axoplasm.

Animals↗
PubMed2000

Olivocochlear efferent vs. middle-ear contributions to the alteration of otoacoustic emissions by contralateral noise.

The medial olivocochlear efferent bundle is the key element of a bilateral efferent reflex activated by sound in either ear and acting directly on cochlear outer hair cells (OHC) via numerous cholinergic synapses. It probably contributes to regulating the mechanical activity of the cochlea. Otoacoustic emissions, being sounds emitted by the cochlea as a reflection of its activity and suppressed by efferent activation, are increasingly considered to be the privileged tool for a noninvasive assessment of the efferent reflex. However, confounding effects on otoacoustic emissions can occur. A primary influence is middle-ear muscle reflex activation, which shares common features with the effects of cochlear efferent activation. We report a systematic comparison of the responses of human otoacoustic emissions to efferent activation by low-level noise in the contralateral ear to various middle-ear manipulations (reflex contractions of the stapedius muscle induced by high-level contralateral noise; moderate middle-ear pressure changes). The profiles of level and phase changes of otoacoustic emissions as a function of frequency were highly specific to the origin of the effects. The changes induced by middle-ear manipulations matched the predictions computed from a standard lumped-element middle-ear model, with one or two peaks around the resonance frequency(ies) of the involved subsystem, stapes or tympanic membrane. In contrast, the efferent effect was completely different, exhibiting a broadband-level suppression associated with a small phase lead. We propose that a careful vector analysis of otoacoustic emission modifications enables the identification of the contribution of the efferent reflex without ambiguity even when it is mixed with middle-ear effects. Thereby, otoacoustic emissions can be used more reliably as noninvasive probes of efferent olivocochlear function.

Acoustic Stimulation↗
PubMed1996

Peripheral retinopathy in offspring of carriers of Norrie disease gene mutations. Possible transplacental effect of abnormal Norrin.

BACKGROUND: The Norrie disease (ND) gene (Xp11.3) (McKusick 310600) consists of one untranslated exon and two exons partially translated as the Norrie disease protein (Norrin). Norrin has sequence homology and computer-predicted tertiary structure of a growth factor containing a cystine knot motif, which affects endothelial cell migration and proliferation. Norrie disease (congenital retinal detachment), X-linked primary retinal dysplasia (congenital retinal fold), and X-linked exudative vitreoretinopathy (congenital macular ectopia) are allelic disorders. METHODS: Blood was drawn for genetic studies from members of two families to test for ND gene mutations. Sixteen unaffected family members were examined ophthalmologically. If any retinal abnormality were identified, fundus photography and fluorescein angiography was performed. RESULTS: Family A had ND (R109stp), and family B had X-linked exudative vitreoretinopathy (R121L). The retinas of 11 offspring of carrier females were examined: three of seven carrier females, three of three otherwise healthy females, and one of one otherwise healthy male had peripheral inner retinal vascular abnormalities. The retinas of five offspring of affected males were examined: none of three carrier females and none of two otherwise healthy males had this peripheral retinal finding. CONCLUSIONS: Peripheral inner retinal vascular abnormalities similar to regressed retinopathy of prematurity were identified in seven offspring of carriers of ND gene mutations in two families. These ophthalmologic findings, especially in four genetically healthy offspring, strongly support the hypothesis that abnormal Norrin may have an adverse transplacental (environmental) effect on normal inner retinal vasculogenesis.

Adult↗
PubMed1999

HIV-1 DNA vaccines.

HIV-1 was among the original DNA vaccine targets and HIV DNA vaccines are now in human trials. Lack of strong correlates of protective immunity makes vaccine design difficult; however, DNA vaccines have the potential to be an ideal vaccine and therapeutic approach against HIV-1. DNA vaccines induce conformational-dependent antibodies, mimic live vaccines but without the pathogenic potential, and can easily be made polyvalent. Genes which encode important CTL and antibody epitopes can be included while those that confer pathogenicity, virulence, antibody enhancement or represent non-conserved epitopes can be excluded. In our hands pre-treatment of muscles with bupivacaine or cardiotoxin did not offer any advantage over no muscle pre-treatment or gene gun inoculation of skin although gene gun immunization seem to favour a Th2 type response. As DNA vaccine candidates we have compared vaccines encoding native HIV MN gp160 with Rev-independent synthetic genes encoding MNgp160 and MNgp120 using mammalian high expression codons. In these experiments the gene encoding secreted gp120 gave highest antibody neutralizing titers. High and fast antibody responses could also be obtained by transferring the HIV-1 MN V3 loop to the secreted HBsAg as a fusion gene vaccine. Thus, in the case of HIV-1 MN genes encoding secreted surface glycoproteins may be preferred instead of membrane bound envelopes. CTL responses were induced in all cases. However, in order to meet the high diversity of HIV and HLA types our approach is to include many CTL epitopes in a multivalent minigene vaccine. We found that gene gun DNA vaccination with minimal epitopes could induce specific CTL. Flanking sequences influenced the CTL response but was not needed. DNA vaccines encoding known and computer predicted CTL epitopes are now being developed.

AIDS Vaccines↗
PubMed2002

Analysis of transcriptomes of human malaria parasite Plasmodium falciparum using full-length enriched library: identification of novel genes and diverse transcription start sites of messenger RNAs.

Now that the sequencing of the complete genome of the human malaria parasite Plasmodium falciparum is now underway, importance of analyses of complementary DNAs (cDNAs) is looming up. We constructed a full-length-enriched cDNA library from erythrocytic stage P. falciparum using the 'oligo-capping' method (Nucleic Acids Res. 29 (2001) 70). In this report we describe the novel genes identified using this library and detailed characterization of transcriptional start site of knob-associated histidine rich protein gene. Contrary to the previous report we conclude all the transcripts of plasmodium genes have diverse start sites. Sequence comparisons between the cDNAs and the complete sequences of chromosomes 2 identified three novel genes that had been missed by computational predictions. Moreover, analysis of transcriptional start sites revealed that the average length of the 5' untranslated region was 346 nt, which is much longer than that in humans. The transcriptional start sites of all the genes studied were far more diverse than those of human genes. These observations may reflect unique mechanism(s) of gene expression in this organism, which has an extremely AT-rich genome.

5' Untranslated Regions↗
PubMed2001

Antibody responses to HIV-1 antigens are higher in HIV-1(+) intravenous drug users than in HIV-1(+) homosexuals.

Immune responses to HIV-1 infection of 42 HIV-1-positive asymptomatic intravenous drug users (IVDUs) were compared with those of 135 HIV-1-infected asymptomatic homosexual men in the present study. Twenty-five HIV-1(-) individuals served as normal controls. The comparison included antibody responses to five computer-predicted epitopes of HIV-1 p17, and viral proteins gp120 and p24 as well as p17. Major immunophenotypes were also investigated. Results showed that antibody responses to the five epitopes were significantly higher in the IVDUs. A larger proportion of the IVDUs, with respect to that of homosexuals, showed positive antibody responses to p24 and p17, respectively. However, the antibody response to gp120 was similar between the two cohorts. Immunophenotyping showed that HIV-1(+) homosexuals had higher profiles in most of the major subsets than did the IVDUs, especially in the total count of lymphocytes, absolute numbers of CD3+ cells and CD8+ cells. It appeared that the HIV-1(+) IVDU cohort had higher antibody responses to most of the viral antigens, but had lower levels of lymphocyte subsets in comparison with HIV(+) homosexuals.

Antibody Formation↗
PubMed1998

A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli.

We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the expression of the virF gene present on the large virulence plasmid of Shigella and enteroinvasive Escherichia coli in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the VirG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of H-NS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30 degrees C. The expression of a virF-lacZ translational fusion in E.coli wild type and in an hns-defective derivative grown at 30 degrees or 37 degrees C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virF expression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controls virF expression by binding to the virF promoter and by repressing its expression at low temperature and at low pH.

Bacterial Proteins↗
PubMed1991

The mature MgPa-adhesin of Mycoplasma genitalium.

A high molecular weight protein of Mycoplasma genitalium (MgPa-protein) was isolated by fractionated solubilization with 1% CHAPS, followed by subsequent extraction with 2% octylglucoside and size exclusion chromatography. The comparison of the N-terminal sequence reported here with published nucleotide sequence data revealed the existence of a signal sequence; the molecular weight of the mature MgPa-protein was calculated to be 153, 134 dalton. The protein shares antigenic determinants with the adhesin of Mycoplasma pneumoniae (P1-protein). Therefore the amino acid sequence of the MgPa-protein was matched to the P1-protein sequence. Five of seven computer predicted hydrophobic regions of both amino acid sequences were located in corresponding regions.

Adhesins, Bacterial↗
PubMedDate not supplied

Present and future in vitro approaches for drug metabolism.

The 1980s through 1990s witnessed the widespread incorporation of in vitro absorption, distribution, metabolism, and excretion (ADME) approaches into drug development by drug companies. This has been exemplified by the integration of the basic science of cytochrome P450s (CYPs) into most drug metabolism departments so that information on the metabolic pathways of drugs and drug-drug interactions (DDIs) is no longer an academic exercise, but essential for regulatory submission. This has come about due to the application of a variety of new technologies and in vitro models. For example, subcellular fractions have been widely used in metabolism studies since the 1960s. The last two decades has seen the increased use of hepatocytes as the reproducibility of cell isolations improved. The 1990s saw the rejuvenation of liver slices (as new slicers were developed) and the utilization of cDNA expressed enzymes as these technologies matured. In addition, there has been considerable interest in extrapolating in vitro data to in vivo for parameters such as absorption, clearance and DDIs. The current philosophy of drug development is moving to a 'fail early--fail cheaply' paradigm. Therefore, in vitro ADME approaches are being applied to drug candidates earlier in development since they are essential for identifying compounds likely to present ADME challenges in the latter stages of drug development. These in vitro tools are also being used earlier in lead optimization biology, in parallel with approaches for optimizing target structure activity relationships, as well as identification of DDI and the involvement of metabolic pathways that demonstrate genetic polymorphisms. This would suggest that the line between discovery and development drug metabolism has blurred. In vitro approaches to ADME are increasingly being linked with high-throughput automation and analysis. Further, if we think of perhaps the fastest available way to screen for successful drugs with optimal ADME characteristics, then we arrive at predictive computational algorithms, which are only now being generated and validated in parallel with in vitro and in vivo methods. In addition, as we increase the number of ADME parameters determined early, the overall amount of data generated for both discovery and development will increase. This will present challenges for the efficient and fast interpretation of such data, as well as incorporation and communication to chemistry, biology, and clinical colleagues. This review will focus on and assess the nature of present in vitro metabolism approaches and indicate how they are likely to develop in the future.

Animals↗
PubMed1998

How and why phosphotyrosine-containing peptides bind to the SH2 and PTB domains.

BACKGROUND: Specific recognition of phosphotyrosine-containing protein segments by Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains plays an important role in intracellular signal transduction. Although many SH2/PTB-domain-containing receptor-peptide complex structures have been solved, little has been done to study the problem computationally. Prediction of the binding geometry and the binding constant of any peptide-protein pair is an extremely important problem. RESULTS: A procedure to predict binding energies of phosphotyrosine-containing peptides with SH2/PTB domains was developed. The average deviation between experimentally measured binding energies and theoretical evaluations was 1.8 kcal/mol. Binding states of unphosphorylated peptides were also predicted reasonably well. Ab initio predictions of binding geometry of fully flexible peptides correctly identified conformations of two pentapeptides and a hexapeptide complexed with a v-Src SH2 domain receptor with root mean square deviations (rmsds) of 0.3 A, 1.2 A and 1.5 A, respectively. CONCLUSIONS: The binding energies of phosphotyrosine-containing complexes can be effectively predicted using the procedure developed here. It was also possible to predict the bound conformations of flexible short peptides correctly from random starting conformations.

Animals↗
PubMed2005

Reengineering the pharmaceutical industry by crash-testing molecules.

The recent decline in drug approvals and the increase in late-stage failures indicate that the ability to generate and screen large numbers of molecules has not improved the drug pipeline. Perhaps the pharmaceutical industry should follow the example of the automotive industry and agree upon a shared modeling language with vendors and academics to enable integration of predictive computational tools across the industry. This will then enable the virtual 'crash-testing' of drugs before synthesis, biological testing and, most importantly, clinical trials. This represents an ambitiously progressive approach using the models for simulating every stage of the drug discovery and development process. Combining the relevant computational algorithms into a grand unified model would enable prioritization of the best ideas before pursuing a discovery program, selecting a target or synthesizing a molecule. The successful application of these virtual crash-testing principles by any of its current proponents could revitalize the pharmaceutical industry so that failure is avoided.

Chemistry, Pharmaceutical↗
PubMed2001

High-throughput docking for lead generation.

Recent improvements in flexible docking technology may lead to a bigger role for computational methods in lead discovery. Although fast and accurate computational prediction of binding affinities for an arbitrary molecule is still beyond the limits of current methods, the docking and screening procedures can select small sets of likely lead candidates from large libraries of either commercially or synthetically available compounds.

Binding Sites↗
PubMed2002

Latest developments in crystallography and structure-based design of protein kinase inhibitors as drug candidates.

Protein kinases have been identified as being implicated in many diseases, and the launch of the anti-cancer Bcr/Abl-kinase inhibitor Glivec has been a major advance in validating protein kinases as 'druggable' targets. High-resolution data exists for many classes of protein kinases and, in some cases, these structures are co-complexed with an inhibitor and/or substrate mimic. Coupled with the increasing reliability of computational predictions, structure-based design is now playing an increasingly important role in the discovery and optimisation of novel, potent and selective protein kinase inhibitors.

Amino Acid Sequence↗
PubMed2000

Cloning and characterization of the TATA-less promoter from the human GFI1 proto-oncogene.

The growth factor independent 1 (GFI1) gene encodes a zinc finger protein which acts as a transcriptional repressor and confers growth factor independence on tumor cells, as suggested by the study of its mouse ortholog, Gfi1. We previously isolated the human GFI1 gene but no information about the structure and location of the promotor of this gene has been reported. In this study we have cloned and characterized the human GFI1 promoter. The nucleotide sequence of the promoter region is GC-rich and does not contain a typical TATA or CAAT box. Several Sp1 sites are present and computer predictions indicate that either of the two Sp1 sites might serve as the sites for transcription initiation. Analysis of various lengths of the promoter region using the luciferase reporter assay identifies a functional promoter that is active in NIH3T3 cells. The strongest activity lies within a region 312-602 basepairs upstream from the translation start site.

3T3 Cells↗
PubMed2026

Selective Macrocyclic WEE1 Kinase Inhibitors with Strong Efficacy against Patient-Derived Colorectal Cancer Organoids.

Macrocyclization can enhance the selectivity of acyclic compounds toward structurally similar biological targets such as kinases. WEE1 regulates cellular homeostasis and is a promising target in oncology. The clinical candidate AZD1775 (1) failed to progress past Phase II trials because of patient tolerability issues, likely due to off-target inhibition of polo-like kinase 1 (PLK1). Herein, a computer-aided drug design approach was conducted to develop a macrocycle based on the 1-WEE1 X-ray cocrystal structure. Significantly enhanced WEE1 inhibitory selectivity over PLK1 was determined for leading macrocycle 2, which also demonstrated broader kinome-wide selectivity. Patient-derived organoids from colorectal cancer (CRC) peritoneal and liver metastases, treated with 2, demonstrated comparably strong or enhanced anticancer efficacy compared to that of 1. Against patient-matched normal colon vs primary CRC organoids, 2 potently and selectively treated CRC, as well as enhanced DNA damage compared to 1. Finally, the X-ray cocrystal structure of 2 bound to WEE1 validated its computationally predicted bioactive binding mode.

Humans↗
PubMed1991

Amino acid sequence of the blue copper protein rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a small blue copper protein isolated from Thiobacillus ferrooxidans. The amino acid sequence of the rusticyanin has been determined by the structural characterization of tryptic and endoproteinase Asp-N peptides with use of amino terminal microsequencing, fast atom bombardment mass spectrometry, and electrospray triple-quadrupole mass spectrometry techniques. Amino acid analysis, carboxy-terminal sequence analysis, and circular dichroism spectroscopy were also performed on the protein. Amino acid sequence identity among rusticyanin and six other small blue copper proteins is apparent only in the limited C-terminal region of each protein bearing three of the four putative copper ligands. A structural model of the rusticyanin is proposed where the protein is principally a beta-barrel comprised of six strands. This model is consistent with the circular dichroism data and computational predictions of the secondary structure of rusticyanin. A feature of the model is the hypothesis that Asp 73 may serve as a fourth copper ligand.

Amino Acid Sequence↗
PubMed2003

Characterization and topology of the membrane domain Nqo10 subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans.

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (Nqo1-Nqo14). Of these, seven subunits (Nqo7, Nqo8, and Nqo10-14) which are equivalent to the mitochondrial DNA-encoded subunits of complex I constitute the membrane segment of the enzyme complex; the remaining subunits make up the peripheral part of the enzyme. We report here on the biochemical characterization and heterologus expression of the Nqo10 subunit. The Nqo10 subunit could not be extracted from the Paracoccus membranes by NaI or alkaline treatment, which is consistent with the presumed membrane localization. By using the maltose-binding protein (MBP) fusion system, the Nqo10 subunit was overexpressed in Escherichia coli. The MBP-fused Nqo10 was expressed in membrane fractions of the host cell and was extractable by Triton X-100. The extracted fusion protein was then isolated by one-step affinity purification through an amylose column. By using immunochemical methods in conjunction with cysteine-scanning mutagenesis and chemical modification techniques, the topology of the Nqo10 subunit expressed in E. coli membranes was determined. The data indicate that the Nqo10 subunit consists of five transmembrane segments with the N- and C-terminal regions facing the periplasmic and cytoplasmic sides of the membrane, respectively. In addition, the data also suggest that the proposed topology of the MBP-fused Nqo10 subunit expressed in E. coli membranes is consistent with that of the Nqo10 subunit in the native Paracoccus membranes. From the experimentally determined topology together with computer prediction programs, a topological model for the Nqo10 subunit is proposed.

Membrane Proteins↗
PubMed1999

Site-directed mutagenesis of a human brain ecto-apyrase: evidence that the E-type ATPases are related to the actin/heat shock 70/sugar kinase superfamily.

On the basis of sequence homologies observed between members of the E-type ATPases and the phosphate binding motifs of the actin/heat shock protein 70/sugar kinase superfamily, a human ecto-apyrase was analyzed by site-directed mutagenesis of conserved amino acids in apyrase conserved regions (ACR) I and IV. The expressed proteins were analyzed to assess the significance of these amino acids. A conserved aspartic acid residue in ACR IV was mutated to alanine, asparagine, and glutamic acid, and the relative activity and Km for ATP and ADP were determined. Mutation of this Asp 219 to Ala or Asn yielded an enzyme severely reduced in ATP hydrolyzing activity (>90%) and completely devoid of ADPase activity, along with a similar extent of inhibition of hydrolysis of other nucleoside di- and triphosphates. Interestingly, mutation of Asp 219 to Glu completely restored the ability of the enzyme to hydrolyze nucleoside triphosphates at levels above that of the wild-type enzyme, while the ability to hydrolyze nucleoside diphosphates was slightly reduced. Mutation of a second conserved aspartic acid in ACR I (Asp 62) and two invariant glycine residues in both ACR I (Gly 64) and ACR IV (Gly 221) also severely disrupted nucleotidase activity. These results demonstrate that the E-type ATPases contain the nucleoside phosphate binding domains present in the actin/heat shock protein/sugar kinase superfamily. Together with analysis of computer-predicted secondary structures, the results suggest that the ecto-ATPases and ecto-apyrases are part of, or closely related to, the actin superfamily of proteins.

Actins↗