Driving forces and current-voltage characteristics of amino acid transport in Riccia fluitans

The complete steady-state I–V relationship of α-aminoisobutyric acid transport across the plasmalemma of rhizoid cells from Riccia fluitans has been measured and analysed with special emphasis on α-aminoisobutyric acid equilibrium and saturation conditions. (A) The electrical data show that: (1) the amino acid-induced electrical current saturates after the addition of the amino acid, regardless of the concentration; (2) a steady state is reached 1–2 h after incubation in α-aminoisobutyric acid, but after less that 5 min in the presence of 1 mM CN⁻; (3) the steady-state I–V characteristic of α-aminoisobutyric acid transport is a sigmoid curve and fairly symmetric in current with respect to the voltage axis; and (4) the equilibrium potential is clearly a function of the amino acid accumulation ratio. It is suggested that the sigmoid curve represents the characteristic of carrier-mediated α-aminoisobutyric acid transport with a voltage-insensitive step, possibly the translocation of the unloaded carrier, rate-limiting. Since under normal conditions the voltage-sensitive rate constant k_oi is much greater than k_io, it is further suggested that the energy to drive this system is put into the transfer of positive charge from outside to the cytoplasm. (B) Accumulation ratios have been determined by inspection of current-voltage data, and additionally by compartmental analysis on green thalli from Riccia fluitans. Both methods give ratios far too low compared with the thermodynamically possible accumulation of about 10⁴. It is suggested that substantial leakages via different non-electrical pathways prevent equilibrium at steady state, and it is concluded that in such leaky systems the thermodynamic equilibrium condition is not suitable for estimating stoichiometries.     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Free radical mechanisms in enzyme reactions

Free radicals are formed in prosthetic groups or amino acid residues of certain enzymes. These free radicals are closely related to the activation process in enzyme catalysis, but their formation does not always result in the formation of substrate free radicals as a product of the enzyme reactions. The role of free radicals in enzyme catalysis is discussed.     

Regulation of voltage-gated Ca channels by lipids

Great skepticism has surrounded the question of whether modulation of voltage-gated Ca²⁺ channels (VGCCs) by the polyunsaturated free fatty acid arachidonic acid (AA) has any physiological basis. Here we synthesize findings from studies of both native and recombinant channels where micromolar concentrations of AA consistently inhibit both native and recombinant activity by stabilizing VGCCs in one or more closed states. Structural requirements for these inhibitory actions include a chain length of at least 18 carbons and multiple double bonds located near the fatty acid's carboxy terminus. Acting at a second site, AA increases the rate of VGCC activation kinetics, and in Ca_V2.2 channels, increases current amplitude. We present evidence that phosphatidylinositol 4,5-bisphosphate (PIP₂), a palmitoylated accessory subunit (β_2a) of VGCCs and AA appear to have overlapping sites of action giving rise to complex channel behavior. Their actions converge in a physiologically relevant manner during muscarinic modulation of VGCCs. We speculate that M₁ muscarinic receptors may stimulate multiple lipases to break down the PIP₂ associated with VGCCs and leave PIP₂'s freed fatty acid tails bound to the channels to confer modulation. This unexpectedly simple scheme gives rise to unanticipated predictions and redirects thinking about lipid regulation of VGCCs.     

Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

The effects of Bombyx mori silk strain and extraction time on the molecular and biological characteristics of sericin

Sericin was extracted from three strains of Thai Bombyx mori silk cocoons (white shell Chul1/1, greenish shell Chul3/2, and yellow shell Chul4/2) by a high-pressure and high-temperature technique. The characteristics of sericin extracted from different fractions (15, 45, and 60 min extraction process) were compared. No differences in amino acid composition were observed among the three fractions. For all silk strains, sericin extracted from a 15-min process presented the highest molecular weight. The biological potential of the different sericin samples as a bioadditive for 3T3 fibroblast cells was assessed. When comparing sericin extracted from three silk strains, sericin fractions extracted from Chul4/2 improved cell proliferation, while sericin from Chul 1/1 activated Type I collagen production to the highest extent. This study allows the natural variability of sericin obtained from different sources and extraction conditions to be addressed and provides clues for the selection of sericin sources.     

Current and emerging commercial optical biosensors.

The field of commercial optical biosensors is rapidly evolving, with new systems and detection methods being developed each year. This review outlines the currently available biosensor hardware and highlights unique features of each platform. Affinity-based biosensor technology, with its high sensitivity, wide versatility and high throughput, is playing a significant role in basic research, pharmaceutical development, and the food and environmental sciences. Likewise, the increasing popularity of biosensors is prompting manufacturers to develop new instrumentation for dedicated applications. We provide a preview of some of the emerging commercial systems that are dedicated to drug discovery, proteomics, clinical diagnostics and routine biomolecular interaction analysis.