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Enhancing high-speed digitization of single-unit neuronal activity on a microcomputer using a hybrid software-hardware technique.

A new data acquisition technique allows a microcomputer simultaneously to digitize spikes at high rates, analyze spike waveforms for computer-based spike separation and manage other control tasks. The technique has two key features: a software scheduling routine written in a high-level language and a hardware analog delay of neuronal signals using simple hardware external to the computer. The technique provides an alternative for real-time data acquisition and can be used on microcomputers without requiring interrupt processing and assembly language programming.

Action Potentials↗

Biogenesis of iron-sulfur proteins in plants.

Iron-sulfur (Fe-S) clusters are ubiquitous prosthetic groups required to sustain fundamental life processes. The assembly of Fe-S clusters and insertion into polypeptides in vivo has recently become an area of intense research. Many of the genes involved are conserved in bacteria, fungi, animals and plants. Plant cells can carry out both photosynthesis and respiration - two processes that require significant amounts of Fe-S proteins. Recent findings now suggest that both plastids and mitochondria are capable of assembling Fe-S proteins using assembly machineries that differ in biochemical properties, genetic make-up and evolutionary origin.

Iron-Sulfur Proteins↗

Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells.

The polymerization of protein subunits into precursor shells empty of DNA is a critical process in the assembly of double-stranded DNA viruses. For the well-characterized icosahedral procapsid of phage P22, coat and scaffolding protein subunits do not assemble separately but, upon mixing, copolymerize into double-shelled procapsids in vitro. The polymerization reaction displays the characteristics of a nucleation limited reaction: a paucity of intermediate assembly states, a critical concentration, and kinetics displaying a lag phase. Partially formed shell intermediates were directly visualized during the growth phase by electron microscopy of the reaction mixture. The morphology of these intermediates suggests that assembly is a highly directed process. The initial rate of this reaction depends on the fifth power of the coat subunit concentration and the second or third power of the scaffolding concentration, suggesting that pentamer of coat protein and dimers or trimers of scaffolding protein, respectively, participate in the rate-limiting step.

Bacteriophage P22↗

Fluorescence delineation of the surfactant microstructures in the CTAB-sOS-H2O catanionic system.

A key feature of amphiphilic molecules is their ability to undergo self-assembly, a process in which a complex hierarchical structure is established without external intervention. Ternary systems consisting of aqueous mixtures of cationic and anionic surfactants exhibit a rich array of self-assembled microstructures such as spherical and rodlike micelles, unilamellar and multilamellar vesicles, planar bilayers, and bicontinuous structures. In general, multiple complementary techniques are required to explore the phase behavior and morphology of aqueous systems of oppositely charged surfactants. As a novel and effective alternative approach, we use fluorescence spectroscopic measurements to examine the microstructures of aqueous cationic/anionic surfactant systems in the dilute surfactant region. In particular, we demonstrate that the polarity-sensitive fluorophore prodan can be used to demarcate the surfactant microstructures of the ternary system of cetyltrimethylammonium bromide, sodium octyl sulfate, and water. As the fluorescence signature of this probe is dependent on the nature of the surfactant aggregates present, our method is a promising new approach to effectively map complex surfactant phase diagrams.

Anions↗

Gene transfer from mitochondrion to nucleus: novel mechanisms for gene activation from Cox2.

The evolutionarily recent transfer of the gene for cytochrome c oxidase subunit 2 (cox2) from the mitochondrion to the nucleus in legumes is shown to have involved novel gene-activation steps. The acquired mitochondrial targeting presequence is bordered by two introns. Characterization of the import of soybean Cox2 indicates that the presequence is cleaved in a three-step process which is independent of assembly. The final processing step takes place only in the mitochondria of legume species, and not in several non-legume plants. The unusually long presequence of 136 amino acids consists of three regions: the first 20 amino acids are required for mitochondrial targeting and can be replaced by another presequence; the central portion of the presequence is required for efficient import of the Cox2 protein into mitochondria; and the last 12 amino acids, derived from the mitochondrially encoded protein, are required for correct maturation of the imported protein. The acquisition of a unique presequence, and the capacity for legume mitochondria to remove this presequence post-import, are considered to be essential adaptations for targeting of Cox2 to the mitochondrion and therefore activation of the transferred gene in the nucleus.

Amino Acid Sequence↗

Mistargeting of B-type lamins at the end of mitosis: implications on cell survival and regulation of lamins A/C expression.

We previously showed that targeting of protein phosphatase 1 (PP1) to the nuclear envelope (NE) by the A-kinase anchoring protein, AKAP149, correlates with nuclear assembly of B-type lamins in vitro. We demonstrate here that failure of AKAP149-mediated assembly of B-type lamins into the nuclear lamina at the end of mitosis is followed by apoptosis, and induces expression of the gene encoding A-type lamins in cells that normally do not express lamins A/C. In HeLa cells, inhibition of PP1 association with the NE mediated by a peptide containing the PP1-binding domain of AKAP149 results in failure of B-type lamins to assemble, and in their rapid caspase-dependent proteolysis. However, assembly of lamins A/C is not affected. Nonetheless, apoptosis follows within hours of nuclear reformation after mitosis. In lymphoid KE37 cells, which do not express lamins A/C, inhibition of B-type lamin assembly triggers rapid synthesis and nuclear assembly of both lamins A and C before apoptosis takes place. The results indicate that nuclear assembly of B-type lamins is essential for cell survival. They also suggest that mistargeting of B-type lamins at the end of mitosis elicits a tentative rescue process to assemble a nuclear lamina in lymphoid cells that normally do not express lamins A/C.

A Kinase Anchor Proteins↗

Inpatient treatment of headache: an evidence-based assessment.

OBJECTIVE: To evaluate inpatient treatment of headache in the United States. PARTICIPANTS: Participants were selected by the meeting chairpersons, Drs. Diamond and Silberstein as well as Dr. Freitag. Criteria for selection included participation in an inpatient treatment program, headache specialists not affiliated with an inpatient treatment program, clinician with interest in headache but not affiliated with a dedicated headache treatment program. The single meeting held in New York was by invitation only. All invitees were provided with opportunity to present their views and participate in the recommendations of the Consortium. The list of participants at the meeting were: Harvey Blumenthal, MD, Roger Cady, MD, James Couch, MD, Seymour Diamond, MD, Frederick G. Freitag, DO, R. Michael Gallagher, DO, Al Lake III, PhD, Richard Lipton, MD, NinanMathew,MD, Alan Rapoport, MD, Jay Rosenberg, MD, Joel Saper, MD, Stephen Silberstein, MD. Funding for theproject was provided by the US Headache Guidelines Consortium Project through the American Academy of Neurology and the National Headache Foundation. EVIDENCE: Each of the authors were part of the writing committee each with specific areas that they were responsible for conducting the literature reviews and submitting a portion of the original document for editing and review. Dr. Freitag coordinated this process and assembled the first draft of the article for circulation and review among the authors and chairpersons. In the absence of class I evidence consensus expert opinion was fundamental to the process. CONCLUSIONS: The treatment of headache is commonly delivered in the outpatient office environment. There exists a group of patients who have been refractive to outpatient treatment and in whom in patient care may be appropriate. The factors that determine the optimal treatment setting, the methodology of the treatment and the results of such treatment have come under increasing scrutiny secondary to the cost of treatment in the hospital. The US Headache Consortium guidelines project in conjunction with the national headache foundation believed that the need existed to objectively examine the issues surrounding inpatient treatment. The epidemiology of chronic headache is important to understand in this context since essentially all patients that are treated in hospital have high frequency headache often associated with significant disability. This severity of illness produces both health care costs but also is responsible for significant impact on businesses and the economy. The method of treatment of headache in the hospital environment can follow several paths based on the accessibility of the resources specific to headache treatment. While there exist these differing paths of treatment only treatment in a dedicated headache treatment unit has been investigated in depth here in the US though reports from other countries that do not have dedicated inpatient treatment lends support to options in headache treatment. Some have suggested that inpatient treatment is not required even in recidivist patients and limited reports have examined aggressive outpatient treatment as an option in headache management. None of the studies reported to date provide class one evidence for efficacy either inpatient or outpatient. Recommendations are made to assess headache treatment in patients with high frequency headaches to garner better scientific evidence for differing treatment approaches.

Adult↗

The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli.

The topology of integral cytoplasmic membrane proteins can be analyzed using alkaline phosphatase fusions by determining which constructs have low and which have high specific activity. We show that in all cases the enzymatic activity is due to the fraction of the alkaline phosphatase moiety of the fusion protein localized to the periplasm. We present evidence that these fusions can also be used to analyze the process of assembly of cytoplasmic proteins into the membrane. The rate of acquisition of protease resistance of the alkaline phosphatase moiety of such hybrid proteins is compared for fusions to periplasmic and cytoplasmic domains. We show that this process, which is assumed to be representative of export of alkaline phosphatase, is significantly slower for fusions to cytoplasmic and certain periplasmic domains than for most periplasmic domains. These results are discussed in the context of the normal assembly of integral membrane proteins.

ATP-Binding Cassette Transporters↗

Trypanosoma brucei 5'ETS A'-cleavage is directed by 3'-adjacent sequences, but not two U3 snoRNA-binding elements, which are all required for subsequent pre-small subunit rRNA processing events.

Trypanosoma brucei pre-rRNA processing commences by cleavage near the 5' end of 5.8 S sequences. The 5' external transcribed spacer (5'ETS) is removed from pre-small subunit (SSU) rRNAs by sequential cleavages at internal A' and A0 sites, and A1 at the 5' end of SSU rRNA. The A' and A0 sites positionally resemble the U3 small nucleolar RNA-dependent, primary pre-rRNA cleavages of vertebrates and yeast, respectively. Uniquely in T. brucei, two U3-crosslinkable 5'ETS sites are essential for SSU rRNA production: site1b is novel in its 3' location to the A' site, and site3 lies upstream of A0 in a position analogous to the yeast U3-binding site. Here, in vivo analysis of mutated 5'ETS sequences shows that sequences 5' to the A' site are not needed for A' cleavage or SSU rRNA production. A' cleavage is linked to, but is not sufficient to trigger, downstream pre-SSU rRNA processing events. These events require an intact 11 nt sequence, 3'-adjacent to A', which directs efficient and accurate A' cleavage. Neither the A' nearby site1b nor the site3 U3-binding elements affect A' processing, yet each is required for A0 and A1 cleavage, and SSU rRNA production. The same U3 3' hinge bases evidently bind a core element, UGUu/gGGU, within site1a and site3; the U3-site1b interaction is less reliant on base-pairing than the U3-site3 interaction. As yeast U3 5' hinge bases pair to 5'ETS sequences, it is clear that distinct U3 hinge regions can interact at both novel and related 5'ETS sites to promote 3'-proximal 5'ETS processing events in diverse organisms. The T. brucei data fit a model wherein processing factors assemble at the 5'ETS site1a to affect A' cleavage and stabilize a U3-site1b complex, which may work in concert with the downstream U3-site3 complex to assist processing events leading to ribosomal SSU production.

Animals↗

Molecular biology of spider silk.

Spider silks are an intriguing family of fibrous proteins due to their highly repetitive primary sequence, their solution properties and their assembly and processing into fibers with remarkable mechanical properties. Current research efforts aimed at understanding and manipulating genes encoding these proteins are helping to gain insight into the relationships between protein sequence, protein assembly and macromolecular properties.

Animals↗

The mouse major histocompatibility complex: some assembly required.

We have assembled a contig of 81 yeast artificial chromosome clones that spans 8 Mb and contains the entire major histocompatibility complex (Mhc) from mouse strain C57BL/6 (H2b), and we are in the process of assembling an Mhc contig of bacterial artificial chromosome (BAC) clones from strain 129 (H2bc), which differs from C57BL/6 in the H2-Q and H2-T regions. The current BAC contig extends from Tapasin to D17Leh89 with gaps in the class II, H2-Q, and distal H2-M regions. Only four BAC clones were required to link the class I genes of the H2-Q and H2-T regions, and no new class I gene was found in the previous gap. The proximal 1 Mb of the H2-M region has been analyzed in detail and is ready for sequencing; it includes 21 class I genes or fragments, at least 14 olfactory receptor-like genes, and a number of non-class I genes that clearly establish a conserved synteny with the class I regions of the human and rat Mhc.

Animals↗

Deletion of the carboxyl-terminal portion of the transit peptide affects processing but not import or assembly of the small subunit of ribulose-1,5-bisphosphate carboxylase.

Import of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase into the chloroplast has been proposed to involve two proteolytic cleavages which convert the 20-kDa precursor (pSSU) into the mature 14-kDa subunit (SSU) via an 18-kDa intermediate. A deletion mutant (PSd48/57) of pSSU which lacks 10 amino acids in a conserved region in the carboxyl-terminal portion of the transit peptide is converted into a series of 16-18-kDa polypeptides in addition to the mature 14-kDa SSU when imported into isolated pea chloroplasts. We examined import and processing of this mutant pSSU to determine whether the 16-18-kDa SSUs undergo further maturation in the chloroplast stroma to yield 14-kDa SSU. The ratio of incorrectly processed to 14-kDa SSU is stable up to 60 min following import. This indicates that processing of PSd48/57 involves a single proteolytic cleavage which occurs during or immediately following transit across the chloroplast envelope. The carboxyl-terminal portion of the transit peptide confers either sequence specificity for the processing protease or provides a three-dimensional structure necessary for consistent cleavage at the mature amino terminus of SSU. Incorrectly processed SSUs were incorporated into the holoenzyme demonstrating that removal of the entire transit sequence is not necessary for assembly of the holoenzyme.

Amino Acid Sequence↗

Chromosome replication in early Xenopus embryos.

Early embryos of X. laevis achieve exceptional rates of DNA replication and chromatin assembly. These processes have been studied by microinjecting DNA templates into eggs or by incubating DNA in egg extracts. The egg is able to regulate replication of injected DNA without requiring specialized DNA sequences. Assembly of DNA into the nucleosome subunits of chromatin involves interaction of DNA with complexes containing two classes of acidic protein, namely nucleoplasmin and N1/N2.

Animals↗

Cross-linking cell surface class II molecules stimulates Ig-mediated B cell antigen processing.

Th cells bind to peptide-class II complexes presented on B cell surfaces. Recent evidence indicates that upon cross-linking, class II molecules transduce signals that modulate a variety of B cell functions. One possible function of class II signaling is to regulate the assembly of processed Ag-class II complexes. Here we show that cross-linking B cell surface class II molecules augments the processing and presentation of an Ek-restricted Ag to a specific T cell hybrid. Significantly, class II cross-linking only affects processing initiated by Ag binding to the surface Ig. The processing of Ag taken up by fluid phase pinocytosis is not affected by class II cross-linking, nor is the presentation of an antigenic peptide that does not require processing. Augmentation of Ag processing is enhanced by treatment of B cells with dibutyryl cAMP, a second messenger in the class II signaling pathway. The cross-linking of class II molecules does not alter the rate or number of Ig molecules internalized or the biosynthesis or expression of Ek molecules. Moreover, changes in the expression of the B7 family of costimulatory molecules or the adhesion molecule LFA-1 (CD11a/CD18) induced by class II cross-linking do not appear to account for the augmentation of processing observed here. Thus, the cross-linking of class II molecules on B cell surfaces selectively stimulates Ig-mediated Ag processing, indicating that a step in this pathway is a target of class II-mediated signaling events.

Animals↗

Autonomous informational stability in connective tissues.

No coherent theories currently explain connective tissue stability (i.e. 'memory') as well as spatial and temporal adaptability in the face of continual flux of its constituents. Furthermore, explanations of stability based exclusively upon DNA raise certain inherent problems, particularly with the spatial concordance of somatic tissues. As an alternative explanation, it is hypothesized that while connective tissue cells produce extracellular protein precursors through DNA-dependent processes, the assembly, location, orientation and configuration of the extracellular macromolecules as well as their degree of cell attachment depend primarily upon local micro-environmental conditions and/or self-organization rather than strictly cellular processes. The resulting extracellular matrix (ECM) serves as a time- and spatially-variable filter about each cell to afford a relatively consistent micro-environment for all similar cells, regardless of the more variable macro-environment. By insuring a consistent set of signals to the cell, the filter provides a non-genetic memory complementary to genetic memory. The half-lives of constituent molecules define the duration of the filter, allowing the filter to adapt to new environmental demands, yet to maintain a consistent milieu for the cell. The cell/matrix construct permits local, self-optimizing, non-deterministic tissue autonomy obviating the need to postulate certain intricate mechanisms coordinating spatial morphology and temporal behavior.

Animals↗

Kinetic stabilities of double, tetra-, and hexarosette hydrogen-bonded assemblies.

A study of the kinetic stabilities of hydrogen-bonded double, tetra-, and hexarosette assemblies, comprising 36, 72, and 108 hydrogen bonds, respectively, is described. The kinetic stabilities are measured using both chiral amplification and racemization experiments. The chiral amplification studies show that solvent polarity and temperature strongly affect the kinetic stabilities of these hydrogen-bonded assemblies. For example, the activation energy for the dissociation of a tetramelamine from a tetrarosette assembly, a process that involves the breakage of 24 hydrogen bonds, was determined at 98.7 +/- 16.6 kJ mol(-1) in chloroform and 172.8 +/- 11.3 kJ mol(-1) in benzene. Moreover, racemization studies with enantiomerically enriched assemblies reveal a strong dependence of the kinetic stability on the number and strength of the hydrogen bonds involved in assembly formation. The half-lives for double, tetra-, and hexarosette assemblies were found to be 8.4 min, 5.5 h, and 150 h in chloroform at 50 degrees C, respectively. For higher generations of these types of assemblies, the kinetic stabilities become so high that they can no longer measured in a direct manner.

Journal Article↗

Heat shock protein 47 in renal scarring.

Heat shock protein 47 (HSP47) is a collagen-binding protein, thought to play an essential mechanistic role in the assembly and processing of procollagens. HSP47 is increasingly being implicated in the pathogenesis of several human and experimental fibrotic diseases. HSP47 could mediate increased accumulation of collagens in the fibrotic mass, possibly by regulating increased assembly of procollagens. Therefore, modulation of HSP47 might be a valuable tool for manipulation of some fibrotic diseases, including renal scarring

Cicatrix↗

The diaphanous-related formin mDia1 controls serum response factor activity through its effects on actin polymerization.

SRF-dependent transcription is regulated by the small GTPase RhoA via its effects on actin dynamics. The diaphanous-related formin (DRF) proteins have been identified as candidate RhoA effectors mediating signaling to SRF. Here we investigate the relationship between SRF activation and actin polymerization by the DRF mDia1. We show that the ability of mDia1 to potentiate SRF activity is strictly correlated with its ability to promote F-actin assembly. Both processes can occur independently of the mDia1 FH1 domain but require sequences in an extended C-terminal region encompassing the conserved FH2 domain. mDia-mediated SRF activation, but not F-actin assembly, can be blocked by a nonpolymerizable actin mutant, placing actin downstream of mDia in the signal pathway. The SRF activation assay was used to identify inactive mDia1 derivatives that inhibit serum- and LPA-induced signaling to SRF. We show that these interfering mutants also block F-actin assembly, whether induced by mDia proteins or extracellular signals. These results identify novel functional elements of mDia1 and show that it regulates SRF activity by inducing depletion of the cellular pool of G-actin.

3T3 Cells↗