Classical Probe Research Articles

Fluorometric high-throughput screening for inhibitors of cytochrome P450.

Rapid screening for cytochrome P450 inhibitors is part of the current paradigm for avoiding development of drugs likely to give clinical pharmacokinetic drug-drug interactions and associated toxicities. We have developed microtiter plate-based, direct, fluorometric assays for the activities of the principal human drug-metabolizing enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, as well as for CYP2A6, which is an important enzyme in environmental toxicology. These assays are rapid and compatible with existing high-throughput assay instrumentation. For CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, the potency of enzyme inhibition (IC50) is consistent regardless of the probe substrate or assay method employed. In contrast, CYP3A4 inhibition for an individual inhibitor shows significant differences in potency (>300-fold) depending on the probe substrate being used. We have investigated these differences through the use of several structurally distinct fluorescent substrates for CYP3A4 and several classical substrate probes (e.g., testosterone, nifedipine, and midazolam), with a panel of known, clinically significant, CYP3A4 inhibitors. The use of multiple probe substrates appears to be needed to characterize the inhibition potential of xenobiotics for CYP3A4.

Analysis of sex chromosome aneuploidy in 41 patients with Turner syndrome: a study of 'hidden' mosaicism.

We performed a genetic study of sex chromosome mosaicism in 41 Turner syndrome patients. The investigation was carried out in four phases: cytogenetics (G-banding), FISH, PCR for SRY in all 41 cases, and sequencing of the SRY gene in the 2 patients with the Y chromosome. The application of classical alpha-satellite probes (CEP-X and CEP-Y), painting probes (WCP-X and WCP-Y) and also XIST, DXZ4 and two subchromosomal painting libraries (SCPL 116 and SCPL102) covering the short and the long arm of the X chromosome, respectively, allowed us to find new mosaic cell lines (mosaicism) in 37 out of 41 patients; only 4 patients were defined as 45,X non-mosaic. The most frequent hidden mosaic was 45,X/46,XX in 32% of the cases; the presence of isochromosomes comprised 25% and markers 5%. The patients who had been previously diagnosed as mosaics displayed a higher complexity in their karyotypes due to the presence of new cell lines. The Y chromosome and the SRY gene were present in blood and ovarian tissue in 2 patients with karyotypes 45,X/46,XY and 45,X/46,X,idic(Ynf). In both patients, the sequencing of the SRY gene confirmed a nucleotide sequence identical to that of a control male. Our results support the hypothesis of 'the necessity of mosaicism for survival', and thus, a mitotic origin for this syndrome.

Hypomethylation of alphoid DNA and classical satellite DNA on chromosome 1, 9, 16 and Y in extraembryonic tissue

The pattern of DNA methylation can be analyzed on methaphase chromosomes with fluorescein labeled antibodies against 5-methylcytosine. In human extraembryonic tissue lower overall intensity of immunofluorescence in centromeric chromosomal regions correspond to hypomethylation of the DNA when compared with normal human lymphocytes. Pericentromeric regions on chromosomes 1,9,16 and heterochromatin on chromosome Y, which reveal lower levels of immunofluorescence, are rich in classical satellite DNA type II and III. In our experiment methylation-sensitive restriction enzymes, alphoid and classical satellite DNA probes specific for chromosomes 1,9,16 and Y were used. Southern blot analysis on cells from extraembryonic tissue revealed different extent of hypomethylation in different chromosomal regions. Our results confirm overall and sequence-specific hypomethylation of DNA in cells from extraembryonic tissue in comparison with somatic cells.

Surface photovoltage measurements in liquids

We present a simple, compact, and robust arrangement for surface photovoltage measurements of free semiconductor surfaces immersed in liquids. It is based on the classical Kelvin probe arrangement, where the semiconductor sample is put in a liquid-containing, electrically insulating vessel, with an optically transparent window, situated between the sample and the Kelvin probe. At the price of permitting relative, rather than absolute, contact potential difference values, this modification enables easy, routine surface photovoltage measurements of semiconductors in any kind of liquid ambient. The validity and efficiency of this approach are demonstrated by surface photovoltage spectra obtained from the p-InP(100) surface in various liquid etchants.

Eleven-dimensional massless superparticles and matrix theory spin-orbit couplings reexamined

The classical probe dynamics of the eleven-dimensional massless superparticles in the background geometry produced by N source M-momenta is investigated in the framework of N-sector DLCQ supergravity. We expand the probe action up to the two fermion terms and find that the fermionic contributions are the spin-orbit couplings, which precisely agree with the matrix theory calculations. We comment on the lack of non-perturbative corrections in the one-loop matrix quantum mechanics effective action and its compatibility with the supergravity analysis.

Free-Radical and Singlet-Oxygen Mobility in Cotton Probed by EPR Spectroscopy

Spin probe EPR experiments using TEMPO and TEMPOL are reported on dry cotton. The spectra are in the slow tumbling region with τrot ∼ 1 ns, the line width is found to be affected by Heisenberg spin exchange and aggregation of the radical. To probe the entire amorphous zone of cotton, ethanol and water are used to deposit the radicals; to probe the fiber surface specifically toluene was used. Analysis shows that in both regions the viscosity is 30 cP and that the fiber surface is an amorphous layer with a depth of approximately 2 nm. The radical motion follows normal liquid theory with a diffusion rate constant of 2 × 108 mol-1 L s-1. A small proportion of the radicals deposit to an area of much higher viscosity. The classical spin probe method is extended via a simple electron spin polarization experiment. This involved irradiation of the sample and analysis of the radical−triplet mechanism polarization of TEMPOL/1O2, which indicates 1O2 diffuses with a similar rate constant.

Non-perturbative membrane spin-orbit couplings in

Membrane source-probe dynamics is investigated in the framework of the finite N-sector DLCQ M-theory compactified on a transverse two-torus for an arbitrary size of the longitudinal dimension. The non-perturbative two-fermion terms in the effective action of the matrix theory, the (2 + 1)-dimensional supetsymmetric Yang-Mills theory, that are related to the four derivative F 4 terms by the supersymmetry transformation are obtained, including the one-loop term and full instanton corrections. On the supergravity side, we compute the classical probe action up to two-fermion terms based on the classical supermembrane formulation in an arbitrary curved background geometry produced by source membranes satisfying the BPS condition; two-fermion terms correspond to the spin-orbit couplings for membranes. We find precise agreement between the two approaches when the background space-time is chosen to be that of the DLCQ M-theory, which is asymptotically locally anti-de Sitter.

Increased chromosomal instability in peripheral lymphocytes and risk of human gliomas.

Brain tumors exhibit considerable chromosome instability (CIN), suggesting that genetic susceptibility may contribute to brain tumorigenesis. To test this hypothesis, in this pilot study, we examined for CIN in short-term lymphocyte cultures from 25 adult glioma patients and 28 age-, sex- and ethnicity-matched healthy controls (all Caucasian). We evaluated CIN by a multicolor fluorescence in situ hybridization assay using two probes: a classic satellite probe for a large heterochromatin breakage-prone region of chromosome 1 and an alpha satellite probe for a smaller region adjacent to the heterochromatin probe. Our results showed a significant increase in the mean number of spontaneous breaks per 1000 cells in glioma patients (mean +/- SD, 2.4+/-0.8) compared with controls (1.4+/-0.9; P < 0.001). By using the median number of breaks per 1000 cells in the controls as the cutoff value, we observed a crude odds ratio (OR) of 8.5 [95% confidence interval (CI) = 2.05-34.9, P < 0.001] for spontaneous breaks and brain tumor risk. After adjustment for age, sex and smoking status, the adjusted OR was 15.3 (95% CI, 2.71-87.8). A significant increase in cells with chromosome 1 aneuploidy (in the form of hyperdiploidy) (P < 0.001) was also observed in the glioma cases, with an adjusted OR of 6.6 (95% CI = 1.5-30, P < 0.05). These findings suggest that CIN can be detected in the peripheral blood lymphocytes of brain tumor patients and may be a marker for identifying individuals at risk.

Very brief exposure to visual distal cues is sufficient for young mice to navigate in the Morris water maze

This set of two experiments investigated the capacities of C57BL/6 mice to integrate visual distal information. In the first study, we submitted mice of two ages (22 and 65 day old) to the place learning version of the Morris water task. After the acquisition phase, the mice were put in one of three conditions: (1) a classical probe test (only the platform was removed); (2) a rotating probe test (the platform was removed and the distal visual cues were rotated from 90°); and (3) a suppressing probe test (both the platform and the cues were removed). The results indicated that whatever their ages mice used preferentially the visual cues to locate the platform. In the second experiment, groups of mice of either age were subjected to 1 of 4 procedures. Separate groups of mice were required to escape onto a platform located in a fixed position either hidden or visible. Following escape, animals were either given unrestricted visual access to the extramaze environment for the duration of the platform interval (60 s, place learning and cue+place learning groups), or denied this opportunity by switching off the room lights (place−lights learning and cue+place−lights learning groups). The results of this second experiment indicate that whatever their age and the procedure used, mice performed equally and exhibited a spatial bias during the probe test. All together, these results suggest that as early as 22 days of age, mice integrate visual distal information especially while on the way to the goal, and this even so this information was not necessary to solve the task.

Identification of 46,XX/46,XY chimerism in an infant with ambiguous genitalia

A variety of molecular methods can aid in the elucidation of complex chromosomal aberrations. We report the case of a newborn infant with ambiguous genitalia born to a 32 year-old G2 P1. Physical exam at delivery showed a vigorous infant with a small phallus with mid-shaft hypospadias. A bifid scrotum was present, with a palpable gonad bilaterally. Abdominal ultrasound showed no female internal structures. Blood was obtained for karyotype and sex determination, and CAM was ruled out. Initial PCR was positive for sry sequences; amelogenin studies done on the same sample showed only X sequences. The preliminary karyotype showed only 46, XX cells. The amelogenin studies were repeated with a longer exposure, and this time in addition to the normal X sequences, very faint amplification of the Y sequences was visible. Final prophase karyotype showed the majority of cells to be 46, XX, however 4 of 22 cells examined showed a 46, XY pattern. Fluorescent in situ hybridization studies, using both whole chromosome paint for the Y chromosome, and a mixture of alpha satellite and classical satellite probes confirmed the presence of XY cells in the culture. The karyotype was 46.XX[18]/46, XY[4].ish (wcpY + ,DYZ1 + ,DYZ3 + ). The infant is doing well at 3 months and is being raised as a male. Good communications and coordination between the cytogenetic and molecular labs in this potentially confusing case allowed for a rapid diagnosis.

Detection of numerical chromosome abnormalities in human spermatozoa by three-color fluorescence in situ hybridization.

Three-color fluorescence in situ hybridization (FISH) was used to detect numerical X, Y, and 17 chromosomal aberrations in human sperm nuclei. Digoxigeninlabeled alpha satellite chromosome X-specific probe DXZ1, biotin-labeled classical satellite chromosome Y-specific probe DYZ1, and biotin plus digoxigenin-labeled alpha satellite chromosome 17-specific probe D17Z1 were simultaneously hybridized to sperm preparations from donors with normal semen (group A) and abnormal semen (group B) characteristics. The proportions of haploid X, Y, 17 and disomy, diploidy of them before and after swim-up were determined. At least 3,000 sperm were analyzed for each sample. Overall, up to 98% of sperm were labeled with the probes, and all statistical analyses were performed using chi 2 tests. A significant difference was observed between group A and group B in frequency of sex chromosome disomy (p < 0.05). In group B, there were significant differences in frequencies of sex chromosomes disomy (p < 0.05) and diploidy (p < 0.01) before to after swim-up. There was no significant difference in frequency of disomy 17 between the 2 groups. In group A and B, the ratios of X- to Y-bearing sperm were 1:1 (neat semen), but in both groups there was a significant increase in Y-bearing sperm after swim-up. The results of this study demonstrated that abnormal semen has sex chromosome disomy more frequently than does normal semen and that portion of sex chromosome disomic and diploid sperm is removed by swim-up, especially for abnormal semen. These findings suggest that we should be careful in using abnormal semen for IVF, especially for ICSI, and should perform swim-up if possible.

Monitoring on-line of extracellular gamma-amino-4-butyric acid using microdialysis coupled to immunosensor analysis

A method is described for the measurement of gamma-amino-4-butyric acid (GABA) within a microdialysis probe using an antibody-linked assay. A monoclonal antibody for GABA provides the specificity of measurement and these antibodies are affixed on a working platinum electrode within the probe. Determination of bound GABA is performed via an indirect assessment of competitive ligand also bound, and conjugated to horseradish peroxidase which is activated and measured as current change. Using this probe directly for extracellular measurements in the somatosensory cortex of sheep compared favorably to the use of this probe on dialysate emerging from a classical microdialysis probe suggesting that it could be used directly in vivo. The probe has a fast response time (>90% of maximum response within 30 s of start of analysis) and high sensitivity (<0.5 μmol/l) and can acquire data every 2 min. It is stable over a period of time in vivo (>48 h) and is regenerable. The probe design offers on-line measurement, with rapid time resolution of substances not amenable to enzyme-based amperometric measurement.

Detection of Intramolecular Associations in Hydrophobically Modified Pectin Derivatives Using Fluorescent Probes

Hydrophobically modified pectin derivatives were prepared by immobilization of long alkyl chains (C12−C18) at various substitution ratios, using two different synthetic pathways, one affording covalent fixation and the other one leading to a mere ionic association. These derivatives display an associative tendency in semidilute as well as in dilute aqueous solutions. This phenomenon, which stems from both intra- and intermolecular interactions between hydrophobic groups, results in the formation of hydrophobic microdomains. The latter can be characterized, especially in the dilute regime, thanks to fluorescence spectroscopy. Fluorescent molecular rotors, as well as pyrene, a classical fluorescence probe of widespread use, witness the variations of the medium polarity. In addition, they can also provide further information, particularly about the local cohesion of the microenvironment of the probe, without performing any complementary experiment, for example, the addition of quenchers together with the fluorescent probe in the polymer solutions. CAC values derived from polarity changes (CACpolarity), using the molecular rotor as well as pyrene as the fluorescence probes, are significantly different from those determined from the cohesion of the microenvironment (CACcohesion), accessible only with the molecular rotor. This latter type of fluorophore may therefore enable us to determine more accurately the actual critical aggregation concentration.

Four parameter data fit for Langmuir probes with nonsaturation of ion current

The four parameter data fit for Langmuir probes is here generalized within the classical single Langmuir probe theory. The data fit accounts for the variation of the Debye sheath thickness of the probe with the applied voltage which results in a nonsaturation of the ion current. The new fitting formula has been applied to the RFX experimental data. With respect to the use of the simple exponential fit, in general a correction of the order of 20% on electron temperature and ion saturation current has been obtained.

Conformal microstrip antenna arrays fed by bentcoplanar waveguides

An experimental investigation of a pyramidal microstrip antenna array fed by coplanar waveguides is presented. The use of a very thin substrate for coplanar lines allows not only a planar design but also simplifies realisation by the bending lines. The measured radiation pattern of this array is similar to that obtained when a classical probe feed is used.

Characterisation of a satellited non-fluorescent Y chromosome (Y[nfqs]) by FISH.

A fetus was prenatally diagnosed as having a Y(nfqs) chromosome which was inherited from the father. With the QFQ technique, the Yqh was observed to be nonfluorescent and contained cytological...

Long-distance interactions ofD-brane bound states and longitudinal five-brane inM(atrix) theory

We discuss long-distance, low-velocity interaction potentials for processes involving longitudinally boosted M5-brane (corresponding in type IIA theory language to the 1/4 supersymmetric bound state of 4-brane and 0-brane). We consider the following scattering configurations: (a) D=11 graviton off longitudinal M5-brane, or, equivalently, 0-branes off marginal 4+0 bound state; (b) M2-brane off longitudinal M5-brane, or a non-marginal 2+0 bound state off marginal 4+0 bound state; (c) two parallel longitudinal M5-branes, or two 4+0 marginal bound states. We demonstrate the equivalence between the classical closed string theory (supergravity) and M(atrix) model (one-loop super Yang-Mills) results for the leading terms in the interaction potentials. The supergravity results are obtained using a generalisation of the classical probe method which allows one to treat bound states of D-branes as probes by introducing non-trivial world-volume gauge field backgrounds.

Predisposing genes and increased chromosome aberrations in lung cancer cigarette smokers

Genotoxic effects linking cigarette smoking with lung cancer have not been consistently demonstrated, therefore claims for the cause–effect relationships are vigorously contested. Using matched populations of 22 lung cancer patients who have been cigarette smokers (LCP), 22 non-cancerous cigarette smokers (SC) and 13 non-smokers (NSC), we have applied the fluorescence in situ hybridization (FISH) tandem probe assay to elucidate the frequency of chromosome breakage among the participants. Two probes were used, a classical satellite probe which hybridizes to the large heterochromatin region of chromosome 1, and an α-satellite probe which targets a small region adjacent to the heterochromatin probe. The highest frequency of structural aberrations was observed in LCP (1.4±0.1) followed by SC (1.25±0.1) and NSC (0.4±0.1). Aberration frequencies were not significantly different between LCP and SC ( p>0.05), however, a statistically significant difference was detected between the smoker populations combined (LCP and SC) and the NSC ( p<0.001). The breakage frequencies showed a positive correlation with duration of smoking for LCP ( r=0.5; p<0.01), but not for SC (P>0.05). In addition, the aberration frequencies were influenced by the inheritance of polymorphic glutathione S-transferase (GST) genes. LCPs missing one or the other GST (GSTM1 or GSTT1) genes were found to have significantly higher chromosome breaks compared to LCPs with both genes present ( p<0.05). Our data indicate that genetic predisposition and chromosome aberrations may be mechanistically related to the initiation of lung carcinogenesis; therefore, they may be useful biomarkers for lung cancer among cigarette smokers.

Interaction of Imidacloprid Metabolites and Analogs with the Nicotinic Acetylcholine Receptor of Mouse Brain in Relation to Toxicity

The favorable selective toxicity of imidacloprid (IMI) to insects versus mammals is attributed to differences in their binding affinity or potency in the nicotinic acetylcholine receptor (nAChR), a proposal tested here by studies on the mechanism of toxicity of IMI metabolites and analogs to mammals. IMI, its desnitro metabolite (DN-IMI), its nitromethylene analog (CH-IMI), and 26 other analogs and metabolites were examined for intraperitoneal toxicity to mice and potency forin vitroinhibition of the binding of [3H]nicotine (the classical nAChR probe) in mouse brain membranes. IMI and 7 analogs with LD50values of 7–50 mg/kg (or intoxication signs at 50 mg/kg) inhibited [3H]nicotine binding by 50% (IC50) at 12–800 nM whereas 21 other analogs that were not toxic at 50 mg/kg gave an IC50of >1000 nM, thereby correlating the toxicity with interaction at the [3H]nicotine binding site. The most potent compounds were DN-IMI and CH-IMI (and its tetrahydropyrimidine analog) with LD50s of 7–24 mg/kg and IC50s of 12–33 nM compared with values for IMI of 39–49 mg/kg and 806 nM, respectively. DN-IMI is therefore a candidate bioactivation product for IMI in mammals. Scatchard analyses indicated that CH-IMIin vitroand possibly DN-IMIin vitroandex vivocompete for the nicotine site (which is at or near the ACh site). When used directly as radioligands, single, saturable, high-affinity binding sites were observed for [3H]DN-IMI (Kd13 nM,Bmax51 fmol/mg protein) and [3H]CH-IMI (Kd16 nM,Bmax20 fmol/mg protein) using the conditions of [3H]nicotine binding (Kd7.8 nM,Bmax87 fmol/mg protein). [3H]DN-IMI also binds to kidney membranes at a site where it is displaced by atropine (ki0.5 μM). [3H]CH-IMI is particularly useful for comparative studies because of high-affinity sites in both insect and mammalian brain.

Gridded retarding pressure sensor for ion and neutral particle analysis in flowing plasmas

Although classical electrostatic plasma probes can provide detailed information regarding electrons and ions, these devices rely on charge-carrying particles both for energy filtering and for detection. Neutral particles are transparent to such diagnostics. In light of this a new probe design was developed to provide a measurement of the neutral particle flux component of a flowing plasma. The simple design adopted for this probe utilizes a set of electrostatic retarding grids to remove the charged particles from the flow. The neutral particles are sensed within the tube of an off-the-shelf hot cathode ionization gauge. Additionally, by varying the potential on the retarding grids, this same probe configuration can be used to measure the ion energy distribution. Preliminary data from this probe were taken in the exhaust plume of a Hall-current accelerator designed for space propulsion. This probe was found to be a very simple, accurate diagnostic tool for such use. By using this probe in combination with interrelated diagnostics the spatial distribution of energetic charge exchange neutrals was quantified in the plume of an SPT-100 thruster.