Differential dynamics of splicing factor SC35 during the cell cycle

Pre-mRNA splicing factors are enriched in nuclear domains termed interchromatin granule clusters or nuclear speckles. During mitosis, nuclear speckles are disassembled by metaphase and reassembled in telophase in structures termed mitotic interchromatin granules (MIGs). We analysed the dynamics of the splicing factor SC35 in interphase and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized in speckles during interphase and dispersed in metaphase. In telophase, GFP-SC35 was highly enriched within telophase nuclei and also detected in MIGs. Fluorescence recovery after photobleaching (FRAP) experiments revealed that the mobility of GFP-SC35 was distinct in different mitotic compartments. Interestingly, the mobility of GFP-SC35 was 3-fold higher in the cytoplasm of metaphase cells compared with interphase speckles, the nucleoplasm or MIGs. Treatment of cells with inhibitors of cyclin-dependent kinases (cdks) caused changes in the organization of nuclear compartments such as nuclear speckles and nucleoli, with corresponding changes in the mobility of GFP-SC35 and GFP-fibrillarin. Our results suggest that the dynamics of SC35 are significantly influenced by the organization of the compartment in which it is localized during the cell cycle

Immunologic glomerulopathies-diagnostic role of immunofluorescence study of renal biopsies

Background: The kidney is a structurally complex organ that has evolved to subserve a number of important functions: excretion of the waste products of metabolism, regulation of body water and salt, maintenance of appropriate acid balance, and secretion of a variety of hormones and autacoids. Some clinical disorders affect more than one structure. In addition, the anatomic interdependence of structures in the kidney implies that damage to one almost always secondarily affects the others. Thus, severe glomerular damage impairs the flow through the peritubular vascular system, conversely, tubular destruction, by increasing interglomerular pressure, may induce glomerular atrophy. Thus, whatever the origin, there is a tendency for all forms of chronic renal disease ultimately to destroy all four components of the kidney, culminating in chronic renal failure and what has been called end-stage contracted kidneys. The functional reserve of the kidney is large, and much damage may occur before functional impairment is evident. The circulating immune complexes play a very major role in various types of glomerular nephropathies.Methods: The present study was conducted on renal biopsies referred to Pathology Department of G.S.V.M. Medical College, Kanpur and Regency Hospital Ltd., Kanpur. Frozen section of renal biopsy was taken for IF studies. Renal biopsy tissue was received in IF fluid containing Ammonium sulphate, N-ethyl malcimide, Magnesium sulphate.Results: The lgG class of immunoglobulins was found to be most fatal to the G13M, the 1gM and IgA were also found to cause glomerular damage. This mechanism was seen responsible for most cases of ICGN. In the present study, fluorescent study of renal biopsy tissue was also done using Hollande’s fixative and it was observed that a better diagnosis could be done when used with routine H & E and immunofluorescent studies.Conclusions: The immunofluorescence microscopy proved to be very useful and essential, for proper diagnosis and therapy of a renal disease. IFM comes out to be a very good indicator of the deposition site and class of immunoglobulin involved in the Immune-complex deposit

Variable-Temperature Time-Resolved Emission Spectra Studies of Random Pyrene Urethane Methacrylate Copolymers with High Pyrene Incorporation

A series of random co-polyurethane methacrylate comb polymers with pyrene (Py) and 3-pentadecylphenol (PDP) as pendant units were prepared by free radical polymerization. The pyrene labeling was varied from 1 to 100 mol %. The excimer emission of these copolymers were studied as a function of both time and temperature using time-resolved emission spectra (TRES) experiments and variable-temperature steady-state fluorescence measurements. Variable-temperature steady-state as well as decay experiments showed that the contribution from excimers via diffusional encounters increased at the cost of pyrene monomer as the temperature increased until ∼50 °C; beyond which nonradiative losses predominated. TRES collected at 25 and 70 °C were compared to study the nature and origin of emitting species as a function of pyrene loading. TRES at 25 °C clearly indicated the presence of ground-state pyrene dimers with emission centered at ∼435 nm which soon gave way to emission centered around 465 and 485 nm in the time-gated spectra collected at higher time intervals. In TRES collected at 70 °C, excimer emission centered at 465 and 485 nm was very high even at short time scales. The lowest pyrene loaded polymer PIHPDP-1Py did not exhibit excimer emission in the TRES collected at 25 °C as well as 70 °C

H‑Bonding vs Non-H-Bonding in 100% Pyrene Methacrylate Comb Polymers: Self-Assembly Probed by Time-Resolved Emission Spectra and Temperature Dependent Fluorescence

The differences in self-organization behavior in novel 100% pyrene labeled comb methacrylate polymers probed as a function of their varied origins of excimer formation are presented. The different structural variations in the polymers included the presence or absence of hydrogen bonding interactions in the form of urethane linkages, short or long alkyl spacer segments separating the pyrene units from the polymer backbone and linear versus kinked urethane linkage. The effect of variable concentration and temperature on the chemical shift of the NH proton of the urethane linkage was probed using ¹H NMR experiments conducted at temperatures varying from 25 to 70 °C at two different concentrations (2.5 and 25 mmol) in DMSO-<i>d</i>₆ as solvent. The photophysical properties of the polymers in dilute DMF solutions were investigated by steady state emission, fluorescence decay studies, time-resolved emission spectra (TRES), and variable temperature emission studies. It was observed that the polymer poly­(PBH) having a non-hydrogen-bondable ester linkage in the pendant chains formed an excimer completely via a static mechanism and the ground state aggregate species were not broken even at higher temperatures. The polymer poly­(PIC) having a short hydrogen-bondable urethane linkage formed an excimer via a static as well as dynamic mechanism. The other hydrogen-bondable urethane methacrylate polymers having a linear linker poly­(PHH) and kinked linker (PIHP) formed excimer mostly via a dynamic mechanism with a very small contribution from the static route. The TRES studies carried out for the polymers provided significant insight into the excimer formation mechanism in these polymers. The variable temperature fluorescence studies highlighted the differences in the H-bonded vs non-H-bonded polymer as a function of their excimer recovery upon cooling

Microstructural Reorganization and Cargo Release in Pyrene Urethane Methacrylate Random Copolymer Hollow Capsules

We report the synthesis of polymer microcapsules by direct one-pot free radical random copolymerization approach. Urethane methacrylate comb monomers having pendant pyrene (Py) and 3-pentadecyl phenol (PDP) units were copolymerized in a random manner using benzoyl peroxide (BPO) as free radical initiator in dimethylformamide (DMF) as solvent. These copolymers and corresponding homopolymers spontaneously self-organized into microspheres upon drop casting from solvents like DMF and tetrahydrofuran (THF). Stable microspheres were obtained in water by dialyzing THF solution of the polymers against water in dialysis bags with molecular weight cutoff of ∼2000. The hollow nature of the spheres was confirmed by rhodamine B (RhB) encapsulation followed by Förster resonance energy transfer (FRET) based fluorescence emission from RhB upon exciting pyrene. The microenvironment inside the capsule was probed by following the <i>I</i>₁/<i>I</i>₃ ratio of pyrene emission as well as RhB release as a function of temperature. The RhB encapsulated in the pyrene homopolymer PIHP-100Py capsules experienced strong donor–acceptor interaction and did not undergo complete release even at high temperature (85 °C). The encapsulated RhB from the copolymers with low pyrene incorporation was released almost fully upon heating beyond 50 °C. Pyrene moieties in the PIHP-100Py were shielded from surrounding water and experienced a hydrophobic environment, whereas in the low pyrene incorporated copolymer the PDP units were better shielded from the hydrophilic environment. This work represents a simple approach to produce polymer hollow capsules, and the varying pyrene incorporation was used to trace the microenvironment inside the capsules

Differential dynamics and stability of lamin A rod domain mutants

Mutations in the human lamin A gene give rise to highly debilitating diseases termed laminopathies. Laminopathic cells harboring certain mutations in lamin A display aberrant nuclear morphology due to abnormal lamina assembly. To understand the molecular mechanisms involved in these processes, we have studied the dynamics and stability of GFP-tagged lamin A constructs harboring disease-causing missense mutations in the rod and tail domains of the protein. Analysis of the mobilities of these proteins by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in intensity after photobleaching (FLIP) techniques in live HeLa cells indicated that mutants that formed large aggregates, like E203G, G232E, Q294P and R386K were substantially more mobile than wild-type and mutant lamins H222P and R482L that assembled at the nuclear periphery. Nuclear extractions with detergent, nucleases and salt resulted in the dispersal of large aggregates into smaller foci throughout the nucleoplasm, whereas more stable lamins were retained at the nuclear periphery. The significant alterations in the dynamics and stability of certain rod domain mutants of lamin A are likely to have profound consequences for the organization of nuclear functions