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PubMed2004

Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase.

BACKGROUND: In the endometrium the steroid hormone progesterone (P), acting through its nuclear receptors, regulates the expression of specific target genes and gene networks required for endometrial maturation. Proper endometrial maturation is considered a requirement for embryo implantation. Endometrial receptivity is a complex process that is spatially and temporally restricted and the identity of genes that regulate receptivity has been pursued by a number of investigators. METHODS: In this study we have used high density oligonucleotide microarrays to screen for changes in mRNA transcript levels between normal proliferative and adequate secretory phases in Rhesus monkey artificial menstrual cycles. Biotinylated cRNA was prepared from day 13 and days 21-23 of the reproductive cycle and transcript levels were compared by hybridization to Affymetrix HG-U95A arrays. RESULTS: Of approximately 12,000 genes profiled, we identified 108 genes that were significantly regulated during the shift from a proliferative to an adequate secretory endometrium. Of these genes, 39 were up-regulated at days 21-23 versus day 13, and 69 were down-regulated. Genes up-regulated in P-dominant tissue included: secretoglobin (uteroglobin), histone 2A, polo-like kinase (PLK), spermidine/spermine acetyltransferase 2 (SAT2), secretory leukocyte protease inhibitor (SLPI) and metallothionein 1G (MT1G), all of which have been previously documented as elevated in the Rhesus monkey or human endometrium during the secretory phase. Genes down-regulated included: transforming growth factor beta-induced (TGFBI or BIGH3), matrix metalloproteinase 11 (stromelysin 3), proenkephalin (PENK), cysteine/glycine-rich protein 2 (CSRP2), collagen type VII alpha 1 (COL7A1), secreted frizzled-related protein 4 (SFRP4), progesterone receptor membrane component 1 (PGRMC1), chemokine (C-X-C) ligand 12 (CXCL12) and biglycan (BGN). In addition, many novel/unknown genes were also identified. Validation of array data was performed by semi-quantitative RT-PCR of two selected up-regulated genes using temporal (cycle day specific) endometrial cDNA populations. This approach confirmed up-regulation of WAP four-disulfide core domain 2 (WFDC2) and SLPI during the expected window of receptivity. CONCLUSION: The identification of P-regulated genes and gene pathways in the primate endometrium is expected to be an important first step in elucidating the cellular processes necessary for the development of a receptive environment for implantation.

Animals↗
PubMed2005

Water mapping in hydrated soft materials.

We present a method based on spatially resolved electron energy-loss spectroscopy in the cryo-STEM to map the spatial distribution of water in frozen-hydrated polymers. The spatial resolution is limited by the dose constraints imposed by radiation damage, and to stay within these constraints, the use of fine electron-probe sizes comes at the cost of reduced counts in the energy-loss spectra. Thus, at the resolution limit, the detection of isolated water-rich pixels or the identification of minor variations in water content across the specimen is complicated because one must distinguish significant fluctuations from noise. Here we develop a criterion with which to guide such a distinction. We characterize the intrinsic noise associated with spectral measurements under given illumination and acquisition conditions. We then use that noise in combination with scatter diagrams to threshold spectrum images and objectively identify statistically significant compositional fluctuations. We illustrate these ideas using a simulated spectrum dataset for a hypothetical blend of hydrophilic and hydrophobic homopolymers. We show that while a direct inspection of the water map may not allow any meaningful conclusions to be drawn, after applying the thresholding approach we can clearly identify the regions of the specimen that are rich in water. We also experimentally study a model blend system comprised of hydrophilic poly(vinyl pyrrolidone) (PVP) dispersed in a hydrophobic matrix of poly(styrene) (PS). By MLS fitting using damaged and undamaged PVP reference spectra, we determine that the critical dose characteristic of dry PVP is approximately 8000 e/nm2 using 200 keV incident electrons. Irradiating frozen-hydrated PVP gives rise to noticeable hydrogen evolution at doses of approximately 1500 e/nm2. To stay within this constraint we use doses of 400 e/nm2 and a pixel spacing in the spectrum imaging of 100 nm. In order to quantitatively map the water, PVP, and PS compositions, we measure their total inelastic scattering cross-sections. Direct inspection of the composition maps reveals the presence of large water-rich domains of the order of approximately 1 microm and the scatter-diagram thresholding approach identifies small water-rich domains one pixel in size.

Frozen Sections↗
PubMed1998

Identification of the basic residues of cytochrome f responsible for electrostatic docking interactions with plastocyanin in vitro: relevance to the electron transfer reaction in vivo.

The prominent basic patch seen in the atomic structure of the lumen-side domain of turnip cytochrome f, consisting of Arg209 and Lys187, 58, 65, and 66, was proposed to be an electrostatically complementary docking site for its physiological electron acceptor, plastocyanin [Martinez, S. E., Huang, D., Szczepaniak, A., Cramer, W. A., and Smith, J. L. (1994) Structure 2, 95-105]. This proposal agrees with solution studies on the cytochrome f/plastocyanin electron-transfer reaction that showed a major contribution of electrostatic interactions to the docking, but not with studies on the oxidation rate of cyt f in vivo using mutants in which the basic patch of cyt f was neutralized. The apparent contradiction might be explained by an unknown electron acceptor protein for cyt f. However, (i) flash-induced oxidation of cyt f is absent in a PC-deficient mutant. (ii) Lys58, 65, and 66 in the large domain and Lys188 and 189 in the small domain are major contributors to the ionic strength dependence of the electron-transfer reaction in solution. Replacement of Lys58 and 65 by neutral residues and of Lys66 by the acidic residue Glu66 resulted in a >10-fold decrease in the rate of electron transfer in solution and complete loss of its ionic strength dependence. Replacement of Lys188 and Lys189 in the small domain of cyt f resulted in a 3-4-fold decrease in the second-order rate constant and a smaller dependence of the overall rate of electron transfer on ionic strength, corresponding to a loss of two positive charges. (iii) Acidification of the thylakoid lumen cannot explain the absence of electrostatic interactions. (iv) Changing the five lysines to acidic residues did not result in any significant retardation of the rate of cyt f oxidation in vivo. If the docking of cyt f and plastocyanin in vivo is mediated by basic residues of cyt f, they are different from those that mediate electron transfer in vitro or that are implicated by simulations of electrostatic interactions of the docking. Alternatively, docking of cyt f/PC in vivo is limited by spatial constraints or release of PC from P700 that precludes a rate-limiting mediation of the cyt f/PC reaction by specific electrostatic interactions. The cyt f/PC system in Chlamydomonas reinhardtii is the first electron-transfer couple for which the role of electrostatics in mediating the docking reaction has been studied both in vitro and in vivo.

Animals↗
PubMed1996

Expression, purification, and characterization of DNA polymerases involved in papovavirus replication.

In recent years, work from a large number of laboratories has greatly expanded our knowledge of the biochemical characteristics and the genetic structure of the DNA polymerases used during papovavirus DNA replication. The development of in vitro DNA replication systems for both SV40 and polyoma virus has been paramount in facilitating the development of the current models describing how DNA polymerase alpha and delta function to replicate the genomes of these two viruses. Our studies have demonstrated that the proteins recognized to be essential for both in vitro SV40 and polyoma viral origin-dependent DNA synthesis can be isolated from cells as an intact complex. We have shown that the human cell MRC closely resembles the murine cell MRC, in both its protein composition and its fractionation and chromatographic profile. In addition, our data regarding both the human and the murine MRC support the dipolymerase model proposed from in vitro DNA replication studies using reconstituted assay systems. In addition, analysis of the nucleotide sequence of the genes encoding DNA polymerase alpha and delta has revealed that the amino acids encoded by several regions of these two genes have been rigorously maintained across evolutionary lines. This information has permitted the identification of protein domains which mediate the complex series of protein-protein interactions that direct the DNA polymerases to the cell nucleus, specify complete or partial exonuclease active sites, and participate in the interaction of each DNA polymerase with the DNA template. Expression studies examining each of the genes encoding DNA polymerase alpha and delta clearly indicate that both DNA polymerases are cell cycle regulated and undergo a dramatic induction in their expression when quiescent cells are stimulated to enter the cell cycle. This is in contrast to the two- to three-fold upregulation in the level of expression of these two genes when cycling cells cross the G1/S boundary. In addition, both proteins are phosphorylated in a cell cycle-dependent manner, and phosphorylation appears to be mediated through the action of a cdc2-dependent protein kinase. Despite all of this new information, much remains to be learned about how papovavirus DNA replication is regulated and how these two DNA polymerases act in vivo to faithfully copy the viral genomes. Studies have yet to be performed which identify all of the cellular factors which potentially mediate papovavirus DNA replication. The reconstituted replication systems have yielded a minimum number of proteins which are required to replicate SV40 and polyoma viral genomes in vitro. However, further studies are needed to identify additional factors which may participate in each step of the initiation, elongation, and termination phases of viral genome replication. As an example, models describing the potential role of cellular helicases, which are components of the MRC isolated from murine and human cells, have yet to be described. It is also conceivable that there are a number of other proteins which serve to attach the MRC to the nuclear matrix, stimulate viral DNA replication, and potentially regulate various aspects of the activity of the MRC throughout viral DNA replication. We are currently working toward characterizing the biochemical composition of the MRC from both murine and human cells. Our goals are to identify all of the structural components of the MRC and to define the role of these components in regulating papovavirus and cellular DNA replication. We have also begun studies to visualize the spatial organization of these protein components within the MRC, examine the regulatory processes controlling the activity of the various components of the MRC, and then develop this information into a coherent picture of the higher order structure of the MRC within the cell nucleus. We believe that this information will enable us to develop an accurate view of the detailed processes mediating both pa

Animals↗
PubMed1997

Switching of gene expression: analysis of the factors that spatially and temporally regulate plant gene expression.

In this chapter, we have reviewed the present research and understanding of several families of transcription factors in plants. From this information, it appears there is good conservation between the types of transcription factors in plants and animals. However, there are several types of factors which have been isolated in plants that remain to be documented in animals (e.g., HD-Zip and GT). These as well as the presence of two types of TATA-binding proteins (TBPs) in plants suggest that although transcription in eukaryotes is highly conserved, fundamental differences may exist. Despite the differences, the modes of regulating transcription are well conserved. Figure 3 summarizes these modes of regulation. In recent years, the role of chromatin structure as well as subcellular localization have been the focus of a vast amount of research in mammals, Drosophila and yeast. However, very little research in these areas has been done in plants. Isolation of genes such as Curly leaf suggest a conservation of genes that influence the formation of heterochromatin-like structures. Whether or not this gene influences chromatin/heterochromatin structure in plants, however, remains to be tested. The study of nuclear localization of factors such as COP1 and KN1 is now leading to models for regulating nuclear transport as well as intercellular transport of transcription factors. Further study of the inter- and intracellular movement of these and other transcription factors may provide information on new modes of regulating transcription. In addition to understanding the role chromatin structure and subcellular localization of transcription factors may have on transcription initiation, the biological role of many plant transcription factors remains to be identified. Several approaches may be taken to understand the mechanisms by which transcription factors influence biochemical and physiological processes in the plant. These steps include 1) identification of the DNA-binding sites of the factors as well as the promoter regions which contain these sites. Presently, this approach is limiting in that not many non-coding regions have been sequenced and characterized in detail. Furthermore, the presence of a putative binding site within a promoter does not necessarily indicate that the factor will bind to the site in vivo. 2) Analysis of the binding affinity for a particular factor to a binding site in comparison to other related factors, via in vitro competition assays and quantitative titrations. This will provide information on how strongly these factors are binding to the sites, but without knowledge of all the factors present in a single cell it is difficult to recreate the in vivo conditions. 3) Generation of transgenic plants or microinjection of DNA/RNA to express a particular factor ectopically, reduce expression of the factor via antisense expression, and creation of dominant negative mutants by overexpression of key dimerization domains may provide information concerning what biological pathways these factors influence. 4) Isolation of mutations in particular transcription factors has been extremely informative in floral development. However, this approach usually entails isolation of a mutant due to a phenotype and eventual mutated locus. The cloning of the locus may or may not involve a transcription factor. 5) Many plant transcription factors have been isolated via sequence similarity to other previously identified and/or characterized transcription factors. However, the biological role of may of these factors is not known. In addition to ectopic expression of these factors by creating transgenic plants, isolation of a loss-of-function mutation may provide valuable information concerning the role of this factor in vivo. Many loss-of-function mutations in MADS box genes have led to a better understanding of how the MADS domain proteins interact with one another as well as how they influence floral development. (ABSTRACT TRUNCATED)

Cell Compartmentation↗
PubMed1992

Identification and developmental expression of a novel low molecular weight neuronal intermediate filament protein expressed in Xenopus laevis.

Xenopus laevis is a valuable model system for the study of vertebrate neuroembryogenesis. However, very few well-characterized nervous system-specific molecular markers are available for studies in this organism. We screened a X. laevis adult brain cDNA library using a cDNA probe for mouse low molecular weight neurofilament protein (NF-L) in order to identify neuron-specific intermediate filament proteins. Clones for two distinct neuron-specific intermediate filament proteins were isolated and sequenced. One of these encoded for a Xenopus NF-L (XNF-L) and the other for a novel neuron-specific Xenopus intermediate filament protein (XNIF) that was present earlier and more abundantly than XNF-L during development. XNIF contained a central rod domain with multiple sequence features characteristic of IF proteins. The XNF-L was very similar to mouse NF-L, with a 77% sequence identity in the rod domain and the presence of a polyglutamic acid region in the tail domain, characteristic of type IV neurofilament proteins. In contrast, XNIF showed only 60% identity to mouse NF-L in the rod domain and lacked the glutamic acid-rich sequence in the tail domain. XNIF also had a very low (approximately 38%) sequence identity in the head and tail domains as compared to NF-L and other neurofilament proteins (45% identity to the head domain of alpha-internexin). In the adult frog, XNIF mRNA is detected by Northern blots only within the nervous system and by in situ hybridization histochemistry exclusively in neurons, particularly in the medullary reticular system and spinal cord. Antisera raised against the unique tail region of XNIF detected a single distinct 60 kDa band in Western blots of nervous system cytoskeletal preparations, and this XNIF immunoreactivity was concentrated in axons in the PNS and in small perikarya in the dorsal root ganglion. In contrast, NF-L immunoreactivity was principally in the large perikarya in the dorsal root ganglion. In development, XNIF mRNA appears more abundant than XNF-L mRNA in all premetamorphic stages examined. XNIF mRNA is first detectable at stage 24 (26 hr), whereas stable expression of XNF-L is at stage 35/36 (50 hr). XNIF immunoreactivity is detectable within the cement gland, within many neuronal cell bodies and axon tracts within the developing nervous system, and within all cellular layers of the developing retina. The availability of these two distinct neuron-specific intermediate filament proteins, with different temporal and spatial expression patterns, should provide new markers as well as targets for functional perturbation in the developing X. laevis nervous system.

Amino Acid Sequence↗
PubMed2005

[Impact of both cardiac-CT and cardiac-MR on the assessment of coronary risk].

Today's definition of coronary artery disease (CAD) comprises two forms: obstructive and non-obstructive CAD. The 31-72% chance of a life-threatening event-like a myocardial infarction-with non-obstructive CAD is well documented in numerous studies. The objective in modern strategies of diagnosis and therapy should therefore be expedient identification of patients at high risk for coronary events, who will benefit from a customized therapy. Before initiating diagnostic procedures of CAD, a well defined strategy should be pursued. There are two possible primary objectives: ASSESSMENT OF THE INDIVIDUAL RISK FOR A CORONARY EVENT: Assessment of the individual "absolute" risk for a coronary event is not possible using single traditional risk factors. The individual risk can be estimated by integrating several of the traditional risk factors into a scoring system. These so-called risk scores (e.g. Framingham score and Procam score), however, have been associated with shortcomings: insufficient discrimination of high-risk from low-risk individuals. The calcium score has therefore become increasingly established; this Agatston score is independent of the traditional risk factors, so there is no correlation between Agatston and Procam scores. Today, the calcium score is considered the superior test for identifying individuals at high risk for a coronary event and its use is recommended by the European Society of Cardiology (ESC) guidelines for prevention of cardiovascular diseases. PROOF OR EXCLUSION OF A HEMODYNAMICALLY SIGNIFICANT CORONARY STENOSIS: Another concept is the definitive proof or exclusion of a hemodynamically "significant" coronary narrowing. The probability of an obstructive CAD is traditionally assessed by the type of chest pain, age, gender and stress-ECG. In patients with a low probability of an obstructive CAD, cardiac catheterization is not indicated, whereas in patients with a high probability of a hemodynamically significant coronary stenosis, an invasive strategy should be performed. Since non-invasive coronary angiography (CTA) with cardiac-CT has been shown to provide a high negative predictive value, CTA (with good imaging quality) is suitable for ruling out a significant obstructive CAD in the group at intermediate risk for an obstructive CAD. Another approach could be a functional test to initially prove a relevant, inducible myocardial ischemia: In a large cohort it was shown that patients will only prognostically benefit from revascularization procedures if the ischemic myocardial area is greater than 10%. Therefore, the assessment of the extent of myocardial ischemia is the domain of modern stress imaging tests. Stress-echocardiography and myocardial scintigraphy have almost the same sensitivity (74-80%, 84-90%, respectively) and specificity (84-89%, 77-86%, respectively), which are considerably higher than for stress-ECG. Cardiac MR is most suitable for the assessment of myocardial perfusion, because it traces the first pass dynamics of gadolinium at rest and during stress in reproducible slices at an acceptable spatial and a high temporal resolution without ionizing radiation. Whether the non-invasive coronary angiography with cardiac-CT and the Adenosin-perfusion imaging with cardiac-MR will completely replace diagnostic cardiac catheterization and stress-echocardiography as well as myocardial scintigraphy remains to be evaluated in further studies.

Coronary Angiography↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗