INVESTIGATION OF EPIDERMAL GROWTH FACTOR RECEPTOR IN LIVE CELLS BY VARIOUS FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY MODALITIES

Ph.DDOCTOR OF PHILOSOPH

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

10.1016/j.bpj.2016.09.049BIOPHYSICAL JOURNAL111102241-225

Homocysteinemia As A Cause For Amaurosis Fugax In A Patient Without An Apparent Embolic Source

Purpose: To report a case with monocular transient vision loss (TVL) associated with Hyperhomocysteinemia. , Methods: We present a case with persistent TVL attacks and high level of homocysteine. , Results: A 32-year-old male had a history of episodes of recurrent monocular TVL. Extensive ophthalmic, systemic and laboratory studies were unremarkable with the exception of high plasma homocysteine level. He never experienced TVL during the 36-month follow-up after starting folate, B12 and B6 except for one episode in which he had discontinued the treatment for three months. , Conclusion: This case may suggest hyperhomocysteinemia as one of the underlying causes of recurrent attacks of TVL without any known source of emboli.PubMe

Case report on the development of a brucellosis-related epidural abscess

Brucellosis is an endemic disease in developing countries. The most commonly observed complications include bone-joint involvement, particularly sacroiliitis and spondylitis. Epidural abscesses caused by brucellosis are a rare complication. We describe the case of a 33-year-old man presenting with high fever, back pain, and weakness. At physical examination, the patient was found to be paraparetic. At thoracic MRI, spondylodiscitis and epidural abscess with significant cord compression were observed. In laboratory examinations, Rose Bengal and tube agglutination tests were positive in patient's serum for brucellosis, and specific antibiotherapy was initiated. Total laminectomy was performed and the abscess was aspirated. The biopsy sample was consistent with chronic non-specific inflammation in acute abscess. No growth was detected in the abscess or blood cultures. Following surgery, medical treatment was initiated and, at six weeks' follow-up, clinical and MRI findings indicated that he had recovered. The diagnosis of spinal epidural abscess due to brucellosis should be considered among differential diagnoses in endemic regions. Early diagnosis and specific treatment are important to prevent later complications

Idiopathic hypereosinophilic syndrome: a case report

Hypereosinophilic syndrome is a spectrum of disorders characterized by marked eosinophilic leukocytosis without identifiable cause or organ dysfunction. We report 37 years old man applied with nonproductive cough and itchy skin eruptions for one year. At the physical examination there were nodular erythematous lesions on his upper extremity. We didn't find any reason which can explain the eosinophily like allergic disease, mycotic infections, parasitic infections, malignancy and collagen tissue diseases with his anamnesis, clinical findings and laboratory tests. He was diagnosed as hypereosinopyhilic syndrome, and treated with hydroxiure. After treatment his skin lesions, leukocytosis and eosinophily were regressed

CENP-C/H/I/K/M/T/W/N/L and hMis12 but not CENP-S/X participate in complex formation in the nucleoplasm of living human interphase cells outside centromeres

<div><p>Kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. Here, we measured the co-migration between protein pairs of the constitutive centromere associated network (CCAN) and hMis12 complexes by fluorescence cross-correlation spectroscopy (FCCS) in the nucleoplasm outside centromeres in living human interphase cells. FCCS is a method that can tell if in living cells two differently fluorescently labelled molecules migrate independently, or co-migrate and thus are part of one and the same soluble complex. We also determined the apparent dissociation constants (K_d) of the hetero-dimers CENP-T/W and CENP-S/X. We measured co-migration between CENP-K and CENP-T as well as between CENP-M and CENP-T but not between CENP-T/W and CENP-S/X. Furthermore, CENP-C co-migrated with CENP-H, and CENP-K with CENP-N as well as with CENP-L. Thus, in the nucleoplasm outside centromeres, a large fraction of the CENP-H/I/K/M proteins interact with CENP-C, CENP-N/L and CENP-T/W but not with CENP-S/X. Our FCCS analysis of the Mis12 complex showed that hMis12, Nsl1, Dsn1 and Nnf1 also form a complex outside centromeres of which at least hMis12 associated with the CENP-C/H/I/K/M/T/W/N/L complex.</p></div

SW-FCCS analysis of CENP-T and CENP-W.

<p>(a): ACF curves of EGFP-(s)-CENP-W (green) and mCherry-(s)-CENP-T (red), and CCF curves (blue, purple) in the nucleoplasm of interphase HEK293 cells. The data show cross-correlation between CENP-T and CENP-W, indicating interaction. (b) and (c): <i>K</i>_<i>d</i> determination using Scatter plot (b) and a histogram (c) of multiple SW-FCCS measurements to determine an effective <i>K</i>_<i>d</i> of the interaction. (d): ACF curves of EGFP-(s)-CENP-T^∆N (green) and mCherry-(s)-CENP-W (red), and CCF curves (blue, purple) in the nucleoplasm of interphase HEK293 cells. The data show reduced cross-correlation between CENP-T^∆N and CENP-W. (e) and (f): <i>K</i>_<i>d</i> determination using Scatter plot (e) and a histogram (f) of multiple SW-FCCS measurements to determine the effective <i>K</i>_<i>d</i> of this interaction between the histone-fold domain of CENP-T (CENP-T^∆N) and CENP-W. A defined interaction is detected by both, the linear fit of the scatter plot as well as the log-normal fit of the histogram.</p

DC-FCCS of EGFP-(s)-CENP-T and mCherry-(s)-CENP-S.

<p>A) Displayed are G versus lag time. Red: FCS- or autocorrelation-curve G (τ) for mCherry, green: FCS- or autocorrelation-curve G (τ) for EGFP, black: cross-correlation-curve G (τ), AC = autocorrelation. CC = cross-correlation, A(AC) = amplitude of autocorrelation curve, A(CC) = amplitude of cross-correlation curve. The cross-correlation analyses are amplified in inserts a. Count rates are displayed over 1 sec (inserts b; green = EGFP and red = mCherry). For the pair EGFP-(s)-CENP-T and mCherry-(s)-CENP-S no indication for complex formation in the nucleoplasm was detected (A(CC)/A(AC_mCherry) = 0%). The cross-correlation analysis (with a magnified scale of G (τ); insert a) resulted in a correlation of 1.001, whereas the autocorrelations yielded 1.322 for EGFP-(s)-CENP-T and 1.106 for mCherry-(s)-CENP-S. This ratio indicates that no nucleoplasmic CENP-T and -S are part of a common complex. B) Localization of cotransfected EGFP-(s)-CENP-T (EGFP) and mCherry-(s)-CENP-S (mCherry) in living human HEp-2 cells which were used for FCCS analysis. White bar = 10 μm. A cell nucleus is displayed showing co-localisation at centromeres (merge) and weak fluorescence in the nucleoplasm. Two locations of the same size and shape, a centromere (spot 1) and the centromere-free position of an FCCS measurement, as shown in Fig 4A (spot 2), in the nucleoplasm were selected for fluorescence intensity analysis. For the analyzed centromere in spot 1 the ratios of nucleoplasmic to centromeric fluorescence intensities was 1:43 for EGFP-(s)-CENP-T and 1:33 for mCherry-(s)-CENP-S. The concentrations of nucleoplasmic proteins, estimated by FCCS, was 6 nM for EGFP-(s)-CENP-T and 14 nM for mCherry-(s)-CENP-S.</p

Cross-correlation by SW-FCCS.

<p>Cross-correlation by SW-FCCS.</p