Fluorescence correlation spectroscopy of the binding of nucleotide excision repair protein XPC-hHr23B with DNA substrates

The interaction of the nucleotide excision repair (NER) protein dimeric complex XPC-hHR23B, which is implicated in the DNA damage recognition step, with three Cy3.5 labeled 90-bp double-stranded DNA substrates (unmodified, with a central unpaired region, and cholesterol modified) and a 90-mer single-strand DNA was investigated in solution by fluorescence correlation spectroscopy. Autocorrelation functions obtained in the presence of an excess of protein show larger diffusion times (τ d) than for free DNA, indicating the presence of DNA-protein bound complexes. The fraction of DNA bound (θ), as a way to describe the percentage of protein bound to DNA, was directly estimated from FCS data. A significantly stronger binding capability for the cholesterol modified substrate (78% DNA bound) than for other double-stranded DNA substrates was observed, while the lowest affinity was found for the single-stranded DNA (27%). This is in accordance with a damage recognition role of the XPC protein. The similar affinity of XPC for undamaged and 'bubble' DNA sub

Applications of raman spectroscopy in dentistry part II: Soft tissue analysis

Raman spectroscopy is rapidly moving from an experimental technique for the analysis of biological molecules to a tool for the real-time clinical diagnosis and in situ evaluation of the oral tissue in medical and dental research. The purpose of this study is to identify various applications of Raman spectroscopy, to evaluate the contemporary status and to explore future directions in the field of dentistry. Several in-depth applications are presented to illustrate Raman spectroscopy in early diagnosis of soft tissue abnormalities. Raman spectroscopy allows to analyze histological and biochemical composition of biological tissues. The technique not only demonstrates its role in the disclosure of dysplasia and malignancy but also in performing guided biopsies, diagnosing sialoliths, and assessment of surgical margins. Raman spectroscopy is used to identify the molecular structures and its components to give substantial information about the chemical structure properties of these molecules. In this paper, we acquaint the utilization of Raman spectroscopy in analyzing the soft tissues in relation to dentistry

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia

The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16+, CD57+ and cytotoxic CD 8+) was studied in the peripheral blood of 18 B-cell chronic lymphocytic leukemia (B-CLL) patients. The absolute numbers of CD 57+, CD 16+ and cytotoxic CD 8+ lymphocytes were increased in the peripheral blood of untreated patients as compared with healthy donors, suggesting a causal relation with the accumulation of malignant B-cells. For 5 B-CLL patients and 5 hematological normal donors, the lymphocyte subpopulations in peripheral blood, lymph nodes and bone marrow were determined. A significant immune response was observed in the lymph nodes of the patients, as reflected by the CD 3+ lymphocytes, which were 1.7–27 times larger in the patients lymph nodes than in their peripheral blood and bone marrow. In contrast, with peripheral blood this was mainly caused by an increase in CD 4+ lymphocytes. The CD 57 lymphocytes in the lymph nodes of the patients had abnormal orthogonal light-scattering signals and an abnormal density of CD 57+ receptors in comparison with their peripheral blood CD 57+ lymphocytes or the CD57+ lymphocytes in the peripheral blood, bone marrow and tonsils of the hematological normal donors. This study shows that although a significant increase of cytotoxic lymphocytes in the peripheral blood of B-CLL patients is observed, the actual distributions of the non-malignant lymphocytes can be quite different at the actual tumor sites, i.e., bone marrow and lymph node

Resonance Raman microspectroscopy of myeloperoxidase and cytochrome b558 in human neutrophilic granulocytes.

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm

Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules

Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the “on–off” blinking observed in the Ser-65 → Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 μs and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is on becomes shorter as the intensity is increased, the off-times are independent of excitation intensity. Statistical analysis of the on- and off-times renders a characteristic off-time of 1.6 ± 0.2 s and allows us to calculate a transition yield of ≈0.5 × 10(−5) from the emissive to the nonemissive state. The saturation excitation intensity at which on- and off-times are equal is ≈1.5 kW/cm(2). On the basis of the single-molecule data we calculate an absorption cross section of 6.5 × 10(−17) cm(2) for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level

Explorations of the application of cyanine dyes for quantitative alpha-synuclein detection

We examined the practical aspects of using fluorescent mono (T-284) and trimethinecyanine (SH-516) dyes for detecting and quantifying fibrillar alpha-synuclein (ASN). We studied the interaction of cyanine dyes with fibrillar proteins using fluorescence spectroscopy and atomic force microscopy. The commercially available classic amyloid stain thioflavin T (Thio T) was used as the reference dye. T-284 and SH-516 dyes can be used for fluorometric quantification of fibrillar wild-type ASN at concentrations of approximately 1.5-20 microg/ml. Both dyes appeared suitable for step-wise monitoring of ASN variants (wild-type and mutants A30P and A53T) aggregation into fibrils in vitro, demonstrating good reproducibility, exceeding that for the commonly used Thio T. Our assay may be used for screening in vitro of agents capable of affecting the aggregation of ASN. In addition, T-284 and SH-516 cyanine dyes were shown to recognize amyloid proteins of various amino acid compositions selectively. T-284 demonstrated particular sensitivity to wild-type and A53T ASN, while for SH 516, the fluorescence response to fibrillar proteins was nearly the same except for lysozymes. T-284 and SH-516 cyanine dyes are sensitive and specific fluorescent probes for monitoring ASN fibril formation process in vitro, quantification of fibrillar ASN in solution, and fluorescent detection of various fibrillar protein species

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA– protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measure ments were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level ∼10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level

Primary neuronal cells express endogenous rαS and neuronal marker MAP2.

<p>A) Immunoblot of cell lysate of primary neuronal cells after 14 days shows immuno-reactivity for MAP2 and αS. B) Cells were fixed and fluorescently labeled; red: phalloidin ^alexa647, cyan: αS ^alexa594, green: anti-MAP2 ^alexa488, blue: DAPI; confocal microscopy; scale bar 10 μm. C) Cells were treated with 100 μM labeled hαS ^alexa488 and unlabeled hαS monomers (ratio 1:3) for six hours and processed as in B; red: phalloidin ^alexa647, green: exogenous hαS ^alexa488, cyan: anti-αS ^alexa594 (endogenous rαS and exogenous hαS), blue: DAPI; confocal microscopy; scale bar 5 μm.</p

hαS interferes with intercellular communication.

<p>Networks of primary cortical rat neurons are electrically stimulated before (day 0) and after administration of 100 μM hαS. A) The first 15 milliseconds after stimulation are dominated by action potentials that were directly triggered by the stimulus pulse (early response; E). These early action potentials are then synaptically propagated through the network and neurons at other electrodes respond up to 300 milliseconds after the stimulation (late response; L). Time progress is visualized by shifting color from blue (day 0) to red (day 5), every line represents the averaged result of 12 stimulation periods (600 stimulations). B) Average development of early and late response after addition of 50 or 100 μM αS, normalized to baseline values. C) Average activity for neural networks treated with 50 μM αS. n = 9 electrodes in three different preparations for each condition.</p

hαS does not aggregate into amyloid fibrils in R12 medium.

<p>A) Fluorescence intensity of the amyloid binding dye ThT as a function of the hαS concentration after incubation in R12 medium for eight days at 37°C (blue) and hαS fibril control sample (red). B) Fluorescence spectra of 1,8-ANS incubated in R12 medium for eight days at 37°C in the presence of 100 μM hαS (red) and without hαS (blue). As a reference the fluorescence spectrum of 1,8-ANS in 10 mM Tris-HCl, pH 7.4 is shown (purple). C) Representative atomic force microscopy (AFM) height image of a 50 μM hαS sample after eight days of incubation in R12 medium indicating that the samples contained small structures. D) In AFM images of the deposited R12 medium with no added hαS similar structures were found as those observed in C). E) Native western blot showing hαS after incubation in 10mM Tris buffer or R12 medium for 0 and 5 days at 37°C.</p