Toroidal multilayer mirrors for laboratory soft X-ray grazing emission X-ray fluorescence.

Efficient soft X-ray spectroscopy in the laboratory is still a challenging task. Here, we report on new toroidal multilayer optics designed and applied with the laser-produced plasma (LPP) source of the Berlin Laboratory for innovative X-ray technologies. The optics are described and characterized, and the application of the updated source to scanning-free grazing emission X-ray fluorescence is demonstrated on thermoelectric gold-doped copper oxide nanofilms.

Quantification of hair cortisol concentration in common marmosets (Callithrix jacchus) and tufted capuchins (Cebus apella).

Quantifying cortisol concentration in hair is a non-invasive biomarker of long-term hypothalamic-pituitary-adrenal (HPA) activation, and thus can provide important information on laboratory animal health. Marmosets (Callithrix jacchus) and capuchins (Cebus apella) are New World primates increasingly used in biomedical and neuroscience research, yet published hair cortisol concentrations for these species are limited. Review of the existing published hair cortisol values from marmosets reveals highly discrepant values and the use of variable techniques for hair collection, processing, and cortisol extraction.

No obvious phenotypic abnormalities in mice lacking the Pate4 gene.

We have previously reported that the hormone calcitonin (CT) negatively regulates bone formation by inhibiting the release of sphingosine-1-phosphate from bone-resorbing osteoclasts. In the context of this study we additionally observed that CT repressed the expression of Pate4, encoding the secreted protein caltrin/Svs7, in osteoclasts from wildtype mice. To assess a possible function of Pate4 in bone remodeling, we utilized commercially available embryonic stem cells with a targeted Pate4 allele to generate Pate4-deficient mice.

Poly-(R)-3-hydroxybutyrates (PHB) are Atherogenic Components of Lipoprotein Lp(a).

The hypothesis is that poly-(R)-3-hydroxybutyrates (PHB), linear polymers of the ketone body, R-3-hydroxybutyrate (R-3HB), are atherogenic components of lipoprotein Lp(a). PHB are universal constituents of biological cells and are thus components of all foods. Medium chain-length PHB (<200 residues) (mPHB) are located in membranes and organelles, and short-chain PHB (<15 residues) are covalently attached to certain proteins (cPHB).

Molecular complementarity between simple, universal molecules and ions limited phenotype space in the precursors of cells.

Background: Fundamental problems faced by the protocells and their modern descendants include how to go from one phenotypic state to another; escape from a basin of attraction in the space of phenotypes; reconcile conflicting growth and survival strategies (and thereby live on 'the scales of equilibria'); and create a coherent, reproducible phenotype from a multitude of constituents.

Presentation Of The Hypothesis: The solutions to these problems are likely to be found with the organic and inorganic molecules and inorganic ions that constituted protocells, which we term SUMIs for Simple Universal Molecules and Ions. These SUMIs probably included polyphosphate (PolyP) as a source of energy and of phosphate; poly-(R)-3-hydroxybutyrate (PHB) as a source of carbon and as a transporter in association with PolyP; polyamines as a source of nitrogen; lipids as precursors of membranes; as well as peptides, nucleic acids, and calcium.

The role of short-chain conjugated poly-(R)-3-hydroxybutyrate (cPHB) in protein folding.

Poly-(R)-3-hydroxybutyrate (PHB), a linear polymer of R-3-hydroxybutyrate (R-3HB), is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight (10,000 to >1,000,000 residues), which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100-200 residues) which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues) which is conjugated to proteins (cPHB), primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins.

Physiological importance of poly-(R)-3-hydroxybutyrates.

Poly-(R)-3-hydroxybutyrates (PHB), linear polymers of (R)-3-hydroxybutyrate, are components of all biological cells in which short polymers (<200 monomer residues) are covalently attached to certain proteins and/or noncovalently associated with polyphosphates - inorganic polyphosphate (polyP), RNA, and DNA. The low concentrations, lack of unusual atoms or functional groups, and flexible backbones of this complexed PHB, referred to as cPHB, make them invisible to many analytical procedures; whereas other physical properties - water-insolubility, high intrinsic viscosity, temperature sensitivity, multiple bonding interactions with other molecules - make them requisite participants in vital physiological processes as well as contributors to the development of certain diseases.

Insights into the structure and assembly of Escherichia coli outer membrane protein A.

Outer membrane protein A (OmpA) of Escherichia coli is a paradigm for the biogenesis of outer membrane proteins; however, the structure and assembly of OmpA have remained controversial. A review of studies to date supports the hypothesis that native OmpA is a single-domain large pore, while a two-domain narrow-pore structure is a folding intermediate or minor conformer. The in vitro refolding of OmpA to the large-pore conformation requires isolation of the protein from outer membranes with retention of an intact disulfide bond followed by sufficient incubation in lipids at temperatures of ≥ 26 °C to overcome the high energy of activation for refolding.

Resolving the native conformation of Escherichia coli OmpA.

The native conformation of the 325-residue outer membrane protein A (OmpA) of Escherichia coli has been a matter of contention. A narrow-pore, two-domain structure has vied with a large-pore, single-domain structure. Our recent studies show that Ser163 and Ser167 of the N-terminal domain (1-170) are modified in the cytoplasm by covalent attachment of oligo-(R)-3-hydroxybutyrates (cOHBs), and further show that these modifications are essential for the N-terminal domain to be incorporated into planar lipid bilayers as narrow pores (≈ 80 pS, 1 m KCl, 22 °C).

Importance of oligo-R-3-hydroxybutyrates to S. lividans KcsA channel structure and function.

In the polyphosphate model of the Streptomyces lividans potassium channel KcsA, four polypeptides, each covalently modified by oligo-(R)-3-hydroxybutyrates (cOHB), surround a core molecule of inorganic polyphosphate (polyP). PolyP attracts, binds, and conducts K(+) in response to an electrochemical stimulus whilst the polypeptides govern access to polyP and regulate its selectivity. However, the role of cOHB has remained uncertain.

Oligo-(R)-3-hydroxybutyrate modification of sorting signal enables pore formation by Escherichia coli OmpA.

The outer membrane protein A (OmpA) of Escherichia coli is a well-known model for protein targeting and protein folding. Wild-type OmpA, isolated either from cytoplasmic inclusion bodies or from outer membranes, forms narrow pores of approximately 80 pS in planar lipid bilayers at room temperature. The pores are well structured with narrow conductance range when OmpA is isolated using lithium dodecyl sulfate (LDS) or RapiGest surfactant but display irregular conductance when OmpA is isolated with urea or guanidine hydrochloride.

[Seasonal dependence of the parameters of quantitative ultrasonic measurements on the peripheral skeleton].

Purpose: To investigate the dependence of parameters of quantitative ultrasound (QUS) on seasonable temperature variation.

Material And Methods: 10 adolescents were examined by QUS at the heel (Sahara, Hologic), at the tibia, at the radius and at the proximal phalanges (Omnisense 7000 P, Sunlight). Measuring took place 0 (T1), 30 (T2), 60 (T3), and 120 (T4) minutes after entering the building at constant room temperature.

Hypothesis: poly-(R)-3-hydroxybutyrate is a major factor in intraocular pressure.

Short chain poly-(R)-3-hydroxybutyrate (cPHB) is a ubiquitous molecule that readily adheres to others, that is covalently added to proteins and that forms, with polyphosphate, ion channels. High levels of cPHB have been implicated in atherosclerosis and in diabetes. Here, we propose a hypothesis in which cPHB adheres to filaments in the extracellular matrix and this raises intraocular pressure.

Role of polyphosphate in regulation of the Streptomyces lividans KcsA channel.

We examine the hypotheses that the Streptomyces lividans potassium channel KcsA is gated at neutral pH by the electrochemical potential, and that its selectivity and conductance are governed at the cytoplasmic face by interactions between the KcsA polypeptides and a core molecule of inorganic polyphosphate (polyP). The four polypeptides of KcsA are postulated to surround the end unit of the polyP molecule with a collar of eight arginines, thereby modulating the negative charge of the polyP end unit and increasing its preference for binding monovalent cations. Here we show that KcsA channels can be activated in planar lipid bilayers at pH 7.

Poly-3-hydroxybutyrate synthase from the periplasm of Escherichia coli.

This is the first report of a poly-3-hydroxybutyrate (PHB) synthase in Escherichia coli. The enzyme was isolated from the periplasm using ammonium sulfate fractionation, hydrophobic, and size-exclusion chromatography and identified by LC/MS/MS as YdcS, a component of a putative ABC transporter. Green Fluorescent Protein-tagged ydcS, purified by 2D native gel electrophoresis, also exhibited PHB synthase activity.

Sorting signal of Escherichia coli OmpA is modified by oligo-(R)-3-hydroxybutyrate.

Escherichia coli outer membrane protein A (OmpA) is a well-established model for the study of membrane assembly. Previous studies have shown that the essential sequence for outer membrane localization, known as the sorting signal, is contained in a segment of the eighth beta-strand, residues 163-171. Sequential digestion of OmpA, purified from outer membranes or inclusion bodies with cyanogen bromide and Staphylococcus aureus GluC, yielded peptides 162-174(LSLGVSYRFGQGE).

Insight into the selectivity and gating functions of Streptomyces lividans KcsA.

Streptomyces lividans KcsA is a 160-aa polypeptide that oligomerizes to form a tetrameric potassium channel. The three-dimensional structure of the polypeptides has been established, but the selectivity and gating functions of the channel remain unclear. It has been shown that the polypeptides copurify with two homopolymers, poly[(R)-3-hydroxybutyrate] (PHB) and inorganic polyphosphate (polyP), which have intrinsic capacities for cation selection and transport.

Infrared and ultraviolet absorptions of matrix isolated C(6)O(2).

In the course of our research into carbon chains trapped in matrices of molecular oxygen, we encountered an IR absorption line at 2180.4 cm(-1), which we tentatively assigned to linear C(6)O(2). In this article, we describe our attempts to confirm the assignment by partial isotopic substitution of carbon by (13)C and oxygen by (18)O.

Haemophilus influenzae outer membrane protein P5 is associated with inorganic polyphosphate and polyhydroxybutyrate.

Outer membrane protein P5 of nontypeable (acapsulate) Haemophilus influenzae (NTHi P5) forms large pores in planar lipid bilayers between symmetric solutions that unpredictably display a nonzero reversal potential. Moreover, NTHi P5 has a high theoretical isoelectric point, calculated as 9.58, which is not in agreement with the experimental isoelectric point, determined as 6.

Pore characteristics of nontypeable Haemophilus influenzae outer membrane protein P5 in planar lipid bilayers.

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5.

Assignment of carbon chain molecules in cryogenic matrices by selective laser-induced oxidation.

We tested and applied a laser-induced oxidation method for identifying the IR-active stretching mode absorptions of linear C(2)(n)(+1) molecules which have known strong UV-vis absorptions. For this purpose, we trapped the molecules of carbon vapor in non-inert matrices (pure O(2) and Ar-O(2) mixtures) at temperatures providing molecular growth. The matrix was exposed to laser light tuned to the wavelength of the UV-vis transition of a specific carbon species.

Kinetics of folding of Escherichia coli OmpA from narrow to large pore conformation in a planar bilayer.

The outer membrane protein of Escherichia coli, OmpA, is currently alleged to adopt two native conformations: a major two-domain conformer in which 171 N-terminal residues form a narrow eight beta-barrel pore and 154 C-terminal residues are in the periplasm and a minor one-domain conformer in which all 325 residues create a large pore. However, recent studies in planar bilayers indicate the conformation of OmpA is temperature-sensitive and that increasing temperature converts narrow pores to large pores. Here we examine the reversibility and kinetics of this transition for single OmpA molecules in planar bilayers of diphytanoylphosphatidylcholine (DPhPC).

A large, voltage-dependent channel, isolated from mitochondria by water-free chloroform extraction.

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria.