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Interdigitated, screen-printed platinum microelectrodes are used for non-Faradaic impedance spectroscopy detection of viable Saccharomyces cerevisiae . The time dependence of the complex impedance of both living and heat-inactivated yeast cells immersed in plain medium or immobilized in agar is measured between 0.01 Hz and 1 MHz for 300 min. To understand the processes probed by the impedance measurement, an equivalent electrical circuit, containing a constant phase element, a charge transfer resistance and a suspension resistance, is established and analyzed. For both heat-inactivated and living yeast cells in plain medium, the suspension resistance increases over time caused by sedimentation of the yeast cells into the stray field of the electrodes. The measured increase is found to be larger for living cells than for heat-inactivated ones. The time dependence of the double-layer capacitance is rather dominated by the metabolic activity of living cells, but a possible impact of cell sedimentation cannot be completely neglected. In experiments carried out with cells immobilized in agar, cell sedimentation is suppressed, and thus, the time evolution of the double-layer capacitance is solely caused by metabolic active cells. Therefore, the time-dependent change of the double-layer capacitance can be used to directly monitor the viability of Saccharomyces cerevisiae in situ , since inactivated cells do not contribute to the signal generation in this case.

Summary Whether gene repositioning to the nuclear periphery during differentiation adds another layer of regulation to gene expression remains controversial. Here, we resolve this by manipulating gene positions through targeting the nuclear envelope transmembrane proteins (NETs) that direct their normal repositioning during myogenesis. Combining transcriptomics with high-resolution DamID mapping of nuclear envelope-genome contacts, we show that three muscle-specific NETs, NET39, Tmem38A, and WFS1, direct specific myogenic genes to the nuclear periphery to facilitate their repression. Retargeting a NET39 fragment to nucleoli correspondingly repositioned a target gene, indicating a direct tethering mechanism. Being able to manipulate gene position independently of other changes in differentiation revealed that repositioning contributes ⅓ to ⅔ of a gene’s normal repression in myogenesis. Together, these NETs affect 37% of all genes changing expression during myogenesis, and their combined knockdown almost completely blocks myotube formation. This unequivocally demonstrates that NET-directed gene repositioning is critical for developmental gene regulation., Graphical Abstract, Highlights • Tissue-specific NETs direct repositioning of critical muscle genes during myogenesis • Expression changes for NET-repositioned genes depend on cell differentiation state • Isolating position from differentiation reveals its contribution to gene expression • Three NETs together affect 37% of all genes normally changing in myogenesis, Muscle-specific nuclear envelope transmembrane proteins (NETs) optimize myogenic gene expression by physically recruiting genes to the periphery and enhancing their repression. Specifically manipulating the position of endogenous genes in myoblasts and myotubes indicates that peripheral localization enhances repression, but only in context of other changes in differentiation.

Impaired cross talk between keratinocytes (KCs) and immune cells is believed to contribute to the pathogenesis of chronic inflammatory skin diseases, such as psoriasis. We have previously identified KCs as a rich source of the serpin protease inhibitor vaspin (serpinA12), originally described as an adipokine in adipose tissue. Herein, we studied whether dysregulated vaspin expression in KCs contributes to the pathogenesis of psoriasis. We found vaspin expression to be closely associated to epidermal differentiation, with low levels in proliferating KCs and high levels in differentiated cells. Consistently, in human psoriasis and in a mouse model of a psoriasis-like skin inflammation, epidermal vaspin expression was significantly down-regulated. Down-regulation of vaspin in KCs resulted in decreased expression of differentiation-associated genes and up-regulation of interferon-inducible and inflammation-associated psoriasis signature genes. Vaspin was also shown to modulate the communication between KCs and inflammatory cells under co-culture conditions. A decrease in vaspin expression in KCs stimulated the secretion of tumor necrosis factor-α, IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 by co-cultured dendritic cells, macrophages, monocytes, and neutrophils. Consequently, the application of vaspin inhibited myeloid cell infiltration in a mouse model of a psoriasis-like skin inflammation. In conclusion, vaspin expression by maturing KCs modulates cutaneous immune responses and may be involved in the pathogenesis of psoriasis.

Human ‘laminopathy’ diseases result from mutations in genes encoding nuclear lamins or nuclear envelope (NE) transmembrane proteins (NETs). These diseases present a seeming paradox: the mutated proteins are widely expressed yet pathology is limited to specific tissues. New findings suggest tissue-specific pathologies arise because these widely expressed proteins act in various complexes that include tissue-specific components. Diverse mechanisms to achieve NE tissue-specificity include tissue-specific regulation of the expression, mRNA splicing, signaling, NE-localization and interactions of potentially hundreds of tissue-specific NETs. New findings suggest these NETs underlie tissue-specific NE roles in cytoskeletal mechanics, cell-cycle regulation, signaling, gene expression and genome organization. This view of the NE as ‘specialized’ in each cell type is important to understand the tissue-specific pathology of NE-linked diseases.

Although its properties have long been used for both typing and prognosis of various tumors, the nuclear envelope (NE) itself and its potential roles in tumorigenesis are only beginning to be understood. Historically viewed as merely a protective barrier, the nuclear envelope is now linked to a wide range of functions. Nuclear membrane proteins connect the nucleus to the cytoskeleton on one side and to chromatin on the other. Several newly identified nuclear envelope functions associated with these connections intersect with cancer pathways. For example, the nuclear envelope could affect genome stability by tethering chromatin. Some nuclear envelope proteins affect cell cycle regulation by directly binding to the master regulator pRb, others by interacting with TGF-ß and Smad signaling cascades, and others by affecting the mitotic spindle. Finally, the NE directly affects cytoskeletal organization and can also influence cell migration in metastasis. In this review we discuss the link between the nuclear envelope and cellular defects that are common in cancer cells, and we show that NE proteins are often aberrantly expressed in tumors. The NE represents a potential reservoir of diagnostic and prognostic markers in cancer.

Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

Novel alginates prepared by independent control of chain stiffness and distribution of G-residues : Structure and gelling properties

Mammalian chromosomes and some specific genes have non-random positions within the nucleus that are tissue-specific and heritable. Work in many organisms has shown that genes at the nuclear periphery tend to be inactive and altering their partitioning to the interior results in their activation. Proteins of the nuclear envelope can recruit chromatin with specific epigenetic marks and can also recruit silencing factors that add new epigenetic modifications to chromatin sequestered at the periphery. Together these findings indicate that the nuclear envelope is a significant epigenetic regulator. The importance of this function is emphasized by observations of aberrant distribution of peripheral heterochromatin in several human diseases linked to mutations in NE proteins. These debilitating inherited diseases range from muscular dystrophies to the premature aging progeroid syndromes and the heterochromatin changes are just one early clue for understanding the molecular details of how they work. The architecture of the nuclear envelope provides a unique environment for epigenetic regulation and as such a great deal of research will be required before we can ascertain the full range of its contributions to epigenetics.

The nuclear lamina, which provides a structural scaffolding for the nuclear envelope, consists largely of a polymer of the intermediate filament lamin proteins. Although different cell types contain distinctive relative amounts of the major lamin subtypes (A, C, B1, and B2), the functions of this variation are not understood. We have investigated the possibility that subtype variation affects lamina stability. We find that homotypic and heterotypic binding interactions of lamin B2 are substantially less resistant to chemical dissociation in vitro than those between the other lamin subtypes, whereas lamin A interactions are the most stable. Surprisingly, removal of the central four-fifths of the rod domain did not substantially weaken the interactions of lamins A and B2, suggesting that other regions also strongly contribute to their binding interactions. In contrast, this rod deletion strongly destabilizes the binding interactions of lamins B1 and C. Consistent with the binding studies, lamins are more readily solubilized by chemical extraction from cells enriched for lamin B2 than from cells enriched for lamin A. This suggests that the distinctive ensemble of heterotypic lamin interactions in a particular cell type affects the stability of the lamin polymer, and, correspondingly, could be relevant to tissue-specific properties of the lamina including its involvement in disease.

AAA proteins remodel other proteins to affect a multitude of biological processes. Their power to remodel substrates must lie in their capacity to couple substrate binding to conformational changes via cycles of nucleotide binding and hydrolysis, but these relationships have not yet been deciphered for any member. We report that when one AAA protein, Hsp104, engages polypeptide at the C-terminal peptide-binding region, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2) drives a conformational change in the middle region. This, in turn, drives ATP hydrolysis in the N-terminal ATPase domain (NBD1). This interdomain communication pathway can be blocked by mutation in the middle region or bypassed by antibodies that bind there, demonstrating the crucial role this region plays in transducing signals from one end of the molecule to the other.

Hsp104 is crucial for stress tolerance in Saccharomyces cerevisiae, and both of its nucleotide-binding domains (NBD1 and NBD2) are required. Here, we characterize the ATPase activity and oligomerization properties of wild-type (WT) Hsp104 and of NBD mutants. In physiological ionic strength buffers (pH 7.5, 37 degreesC) WT Hsp104 exhibits Michaelis-Menten kinetics between 0.5 and 25 mM ATP (Km approximately 5 mM, Vmax approximately 2 nmol min-1 microg-1). ATPase activity is strongly influenced by factors that vary with cell stress (e.g. temperature, pH, and ADP). Mutations in the P-loop of NBD1 (G217V or K218T) severely reduce ATP hydrolysis but have little effect on oligomerization. Analogous mutations in NBD2 (G619V or K620T) have smaller effects on ATPase activity but impair oligomerization. The opposite relationship was reported for another member of the HSP100 protein family, the Escherichia coli ClpA protein, in studies employing lower ionic strength buffers. In such buffers, the Km of WT Hsp104 for ATP hydrolysis decreased 10-fold and its stability under stress conditions increased, but the effects of the NBD mutations on ATPase activity and oligomerization remained opposite to those of ClpA. Either the functions of the two NBDs in ClpA and Hsp104 have been reversed or both contribute to ATP hydrolysis and oligomerization in a complex manner that can be idiosyncratically affected by such mutations.

The [PSI+] factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in β sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a “protein-only” seeded polymerization model for the inheritance of [PSI+].

The HSP100/Clp proteins are a newly discovered family with a great diversity of functions, such as increased tolerance to high temperatures, promotion of proteolysis of specific cellular substrates and regulation of transcription. HSP100/Clp proteins are also synthesized in a variety of specific patterns and, in eukaryotes, are localized to different subcellular compartments. Recent data suggest that a common ability to disassemble higherorder protein structures and aggregates unifies the molecular functions of this diverse family.

In eukaryotic cells, nuclear envelope transmembrane proteins (NETs) are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM). Quantitative determination of the distribution of NETs on the ONM and INM is limited in available approaches, which moreover provide no information about translocation rates in the two membranes. Furthermore, commonly used techniques, such as electron microscopy, are time-consuming, and performed in vitro. Here, we demonstrate a single-point FRAP microscopy technique that enables determination of distribution and translocation rates in vivo with

Human herpesvirus 6 (HHV-6) is a newly identified lymphotropic herpesvirus. We have analyzed viral and host DNA replication in peripheral blood lymphocytes infected in the absence of drugs or infected in the presence of phosphonoacetic acid (PAA) or acyclovir (ACV). The results revealed the following: (i) Infection with HHV-6 resulted in the shutoff of host DNA replication. (ii) PAA at concentrations of 100 and 300 micrograms/ml significantly reduced virus replication. The drug inhibited viral DNA replication, whereas host cell DNA replication was not affected. This strongly suggests that HHV-6 encodes a PAA sensitive viral DNA polymerase. (iii) ACV at 20 microM did not interfere with virus production and virus spread. ACV at 100 microM only partly interfered with virus replication, whereas at 400 microM the block was more complete. Viral DNA replication was not affected by ACV at 20 microM. However, approximately 60 and 85% inhibition in viral DNA replication was observed in the presence of 100 and 400 microM of ACV. (iv) Assays for viral thymidine kinase (TK) revealed no significant increase in TK activity, whereas increased TK activity was noted following infection of the same peripheral blood lymphocytes with herpes simplex virus. Thus, either HHV-6 does not encode a tk enzyme which can phosphorylate ACV or the inefficient block may reflect lower sensitivity of the HHV-6 DNA polymerase to the drug.

Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights.