Changes in Organization of the Endoplasmic Reticulum during Xenopus Oocyte Maturation and ActivationV⃞

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releaseability of Ca2+ by IP3. The clusters dispersed during the Ca2+ wave at activation. Possible relationships of ER structure and Ca2+ regulation are discussed.

Accumulation of Mutant Huntingtin Fragments in Aggresome-like Inclusion Bodies as a Result of Insufficient Protein Degradation

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin–proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14–3-3, and α-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin–proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

Interaction of cyclooxygenases with an apoptosis- and autoimmunity-associated protein.

Cyclooxygenases (COXs) 1 and 2 are 72-kDa, intralumenal residents of the endoplasmic reticulum (ER) and nuclear envelope, where they catalyze the rate-limiting steps in the conversion of arachidonate to the physiologically dynamic prostanoids. Recent studies, including the generation of knockout mice, show COX-1 and COX-2 to have biologically distinct roles within cells and organisms. Also apparent is that arachidonate substrate is selectably metabolized by COX-2 after mitogen stimulation in many cells that contain both isoforms. Because COX-1 and COX-2 are highly conserved in all residues needed for catalysis and in their purified forms have almost identical kinetic properties, we have searched for COX-interacting ER proteins that might mediate these unique isoenzymic properties. Using COXs as bait in the yeast two-hybrid system, we identified autoimmunity- and apoptosis-associated nucleobindin (Nuc) as a protein that specifically interacts with both isoenzymes. COX-Nuc binding was substantiated by immunoprecipitation experiments, which showed that COX-1 and, to a lesser extent, COX-2 form complexes with Nuc in vitro. When overexpressed in COS-1 cells, Nuc was found to be extracellularly released. However, when Nuc was co-overexpressed with COX-1 or COX-2, its release was reduced by >80%. This finding suggests that COX isoenzymes participate in the retention of Nuc within the lumen of the ER, where COX may regulate the release of Nuc from the cell. It also identifies Nuc as a potential regulator of COXs through this interaction.

An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor.

We have identified an amino acid sequence in the Drosophila Transformer (Tra) protein that is capable of directing a heterologous protein to nuclear speckles, regions of the nucleus previously shown to contain high concentrations of spliceosomal small nuclear RNAs and splicing factors. This sequence contains a nucleoplasmin-like bipartite nuclear localization signal (NLS) and a repeating arginine/serine (RS) dipeptide sequence adjacent to a short stretch of basic amino acids. Sequence comparisons from a number of other splicing factors that colocalize to nuclear speckles reveal the presence of one or more copies of this motif. We propose a two-step subnuclear localization mechanism for splicing factors. The first step is transport across the nuclear envelope via the nucleoplasmin-like NLS, while the second step is association with components in the speckled domain via the RS dipeptide sequence.

Protein kinase C chimeras: catalytic domains of alpha and beta II protein kinase C contain determinants for isotype-specific function.

Protein kinase C (PKC) is involved in the proliferation and differentiation of many cell types. In human erythroleukemia (K-562) cells, the PKC isoforms alpha and beta II play distinct functional roles. alpha PKC is involved in phorbol 12-myristate 13-acetate-induced cytostasis and megakaryocytic differentiation, whereas beta II PKC is required for proliferation. To identify regions within alpha and beta II PKC that allow participation in these divergent pathways, we constructed chimeras in which the regulatory and catalytic domains of alpha and beta II PKC were exchanged. These PKC chimeras can be stably expressed, exhibit enzymatic properties similar to native alpha and beta II PKC in vitro, and participate in alpha and beta II PKC isotype-specific pathways in K-562 cells. Expression of the beta/alpha PKC chimera induces cytostasis in the same manner as overexpression of wild-type alpha PKC. In contrast, the alpha/beta II PKC chimera, like wild-type beta II PKC, selectively translocates to the nucleus and leads to increased phosphorylation of the nuclear envelope polypeptide lamin B in response to bryostatin-1. Therefore, the catalytic domains of alpha and beta II PKC contain determinants important for alpha and beta II PKC isotype function. These results suggest that the catalytic domain represents a potential target for modulating PKC isotype activity in vivo.

Basal body-associated DNA: in situ studies in Chlamydomonas reinhardtii.

We have explored the localization of the uni chromosome (LG XIX) of Chlamydomonas reinhardtii using the technique of in situ hybridization. Using standardized methods of cell fixation together with large chromosome-specific probes we have studied the position of uni DNA sequences in metaphase and interphase cells. We find that in dividing cells uni probes identify a condensed metaphase chromosome that shows no specialized orientation. In interphase cells uni hybridization signals occur on the anterior edge of the nucleus at a position where basal bodies are normally associated with the nuclear envelope. These data reveal an underlying spatial organization of uni chromosomal DNA within the interphase nucleus that may be significant in terms of the fact that this chromosome encodes numerous functions affecting basal body and flagellar assembly.

The impact of prolonged fasting during aestivation on the structure of the small intestine in the green-striped burrowing frog, Cyclorana alboguttata

The effects of short-term fasting and prolonged fasting during aestivation on the morphology of the proximal small intestine and associated organs were investigated in the green-striped burrowing frog, Cyclorana alboguttata (Anura: Hylidae). Animals were fasted for 1 week while active or for 3-9 months during aestivation. Short-duration fasting (1 week) had little effect on the morphology of the small intestine, whilst prolonged fasting during aestivation induced marked enteropathy including reductions in intestinal mass, length and diameter, longitudinal fold height and tunica muscularis thickness. Enterocyte morphology was also affected markedly by prolonged fasting: enterocyte cross-sectional area and microvillous height were reduced during aestivation, intercellular spaces were visibly reduced and the prevalence of lymphocytes amongst enterocytes was increased. Mitochondria and nuclei were also affected by 9 months of aestivation with major disruptions to mitochondrial cristae and increased clumping of nuclear material and increased infolding of the nuclear envelope. The present study demonstrates that the intestine of an aestivating frog responds to prolonged food deprivation during aestivation by reducing in size, presumably to reduce the energy expenditure of the organ.

LAP1 interactome and its functional features throughout spermatogenesis

The lamina-associated polypeptide 1 (LAP1) is a type II transmembrane protein of the inner nuclear membrane encoded by the human gene TOR1AIP1. LAP1 is involved in maintaining the nuclear envelope structure and appears be involved in the positioning of lamins and chromatin. In the nuclear envelope, LAP1 is suggested to exist as a complex with A-type and B-type lamins, torsins and emerin. The presence of such complexes suggests that LAP1 may cooperate functionally with these proteins in tissues where they play a critical role. Therefore, the identification of LAP1 binding partners and the signalling pathways where LAP1 participates, is crucial for a better understanding of LAP1 functions. The work described in this thesis addresses novel human LAP1 associated proteins found through bioinformatic tools. Public databases allowed for the discovery of the LAP1 interactome, which was manually curated, identifying several functionally relevant proteins. Subsequently, the integration of multiple bioinformatic tools established novel functions to LAP1 such as DNA damage response and telomere association. In conjunction, bioinformatic results also reinforced the association of LAP1 with mitosis, and the already identified role of LAP1 in nuclear morphology. Interestingly, this association of LAP1 with the regulation of the nuclear envelope structure and mitosis progression, shares functional elements with spermatogenesis. Therefore, this work additionally described the localization of LAP1 and some of its interactors throughout the spermatogenic cycle, in mouse and human testis. The results established that the activity of LAP1 during the mouse spermatogenic cycle is most evident from stage VIII until the end of spermiogenesis, which is characteristic of manchette development. Concomitantly, some LAP1 interactors studied in this work share a similar localization, namely, PP1γ2, Lamin B1 and Lamin A/C. The results obtained from the study of LAP1 throughout different periods of the male reproductive system attributed potential new biological functions to LAP1. Thereby, this work can be the foundation of future studies regarding LAP1 and the regulation of multiple cellular processes and disease conditions.

On the theory of mixing-frequency electron spin-echo envelope modulation spectroscopy

It has recently been stated that the parametrization of the time variables in the one-dimensional (I-D) mixing-frequency electron spin-echo envelope modulation (MIF-ESEEM) experiment is incorrect and hence the wrong frequencies for correlated nuclear transitions are predicted. This paper is a direct response to such a claim, its purpose being to show that the parametrization in land 2-D MIF-ESEEM experiments possesses the same form as that used in other 4-pulse incrementation schemes and predicts the same correlation frequencies. We show that the parametrization represents a shearing transformation of the 2-D time-domain and relate the resulting frequency domain spectrum to the HYSCORE spectrum in terms of a skew-projection. It is emphasized that the parametrization of the time-domain variables may be chosen arbitrarily and affects neither the computation of the correct nuclear frequencies nor the resulting resolution. The usefulness or otherwise of the MIF parameters \gamma\ > 1 is addressed, together with the validity of the original claims of the authors with respect to resolution enhancement in cases of purely homogeneous and inhomogeneous broadening. Numerical simulations are provided to illustrate the main points.

Characterization of mouse profilaggrin: Evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N-terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal anti-body raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Estudos estruturais em ferredoxinas por ressonância magnética nuclear

Dissertação apresentada para obtenção do Grau de Doutor em Química, especialidade Química Inorgânica, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation

The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.

Emergent Antiferromagnetism Out Of The Hidden-order" State In Uru2si2: High Magnetic Field Nuclear Magnetic Resonance To 40 T."

Very high field (29)Si-NMR measurements using a fully (29)Si-enriched URu(2)Si(2) single crystal were carried out in order to microscopically investigate the hidden order (HO) state and adjacent magnetic phases in the high field limit. At the lowest measured temperature of 0.4 K, a clear anomaly reflecting a Fermi surface instability near 22 T inside the HO state is detected by the (29)Si shift, (29)K(c). Moreover, a strong enhancement of (29)K(c) develops near a critical field H(c) ≃ 35.6 T, and the ^{29}Si-NMR signal disappears suddenly at H(c), indicating the total suppression of the HO state. Nevertheless, a weak and shifted (29)Si-NMR signal reappears for fields higher than H(c) at 4.2 K, providing evidence for a magnetic structure within the magnetic phase caused by the Ising-type anisotropy of the uranium ordered moments.

Observation Of D0 Meson Nuclear Modifications In Au+au Collisions At Sqrt[s(nn)] = 200 Gev.

We report the first measurement of charmed-hadron (D(0)) production via the hadronic decay channel (D(0) → K(-) + π(+)) in Au+Au collisions at sqrt[s(NN)] = 200 GeV with the STAR experiment. The charm production cross section per nucleon-nucleon collision at midrapidity scales with the number of binary collisions, N(bin), from p+p to central Au+Au collisions. The D(0) meson yields in central Au + Au collisions are strongly suppressed compared to those in p+p scaled by N(bin), for transverse momenta p(T) > 3 GeV/c, demonstrating significant energy loss of charm quarks in the hot and dense medium. An enhancement at intermediate p(T) is also observed. Model calculations including strong charm-medium interactions and coalescence hadronization describe our measurements.

Espectroscopia de Ressonância Magnética Nuclear de 13C no estudo de rotas biossintéticas de produtos naturais

During the last five decades, as a result of an interaction between natural product chemistry, synthetic organic chemistry, molecular biology and spectroscopy, scientists reached an extraordinary level of comprehension about the natural processes by which living organisms build up complex molecules. In this context, 13C nuclear magnetic resonance spectroscopy, allied with isotopic labeling, played a determinant role. Nowadays, the widespread use of modern NMR techniques allows an even more detailed picture of the biochemical steps by accurate manipulation of the atomic nuclei. This article focuses on the development of such techniques and their impact on biosynthetic studies.