Hydrobenzoin-based rigid chiral polymer

We prepared a rigid, chiral polymer (1) from optically active hydrobenzoin-based subunits. Nonracemic monomer units 6 and 8 were prepared by asymmetric dihydroxylation (AD) methodology and polymerization was carried out under Sonagashira coupling conditions. Polymer 1 was obtained in good yield with a molecular weight M(n) = 5,100 (PDI = 2.3). Modeling suggests that polymer 1 could form a stable helical mainchain conformation in solution or the solid state. The chiroptical data of the polymer and a low-molecular weight model compound (9) are compared.     

Hormone trajectories leading to human birth

The mechanisms regulating human parturition and labor remain unknown. This ignorance is expensive as preterm birth is responsible for 70% of neonatal mortality and 50% of cerebral palsy. Methods for the prediction of preterm birth and treatments for women in preterm labor have poor efficacy reflecting our limited knowledge of the mechanisms involved. Recent research has supported the view that parturition is a cascade of events that commences early in pregnancy and involves the mother, fetus, placenta, membranes, cervix and myometrium. Although a number of the key hormones and proteins involved have been identified, the relationships between these factors in time and tissues remain unclear. Placental production of Corticotropin-releasing hormone (CRH) is proposed as an early event regulating the cascade of events. Central to the onset of parturition will be a mechanism for progesterone withdrawal and estrogen activation in human. Two forms of progesterone receptor with opposing actions exist in the human myometrium. Progesterone receptor A (PR-A) is a dominant negative repressor of progesterone receptor B (PR-B). Preliminary studies strongly support the hypothesis that the onset of human parturition is initiated by rising concentrations of PR-A in the myometrium.     

Two populations of node monocilia initiate left-right asymmetry in the mouse

The vertebrate body plan has conserved handed left-right (LR) asymmetry that is manifested in the heart, lungs, and gut. Leftward flow of extracellular fluid at the node (nodal flow) is critical for normal LR axis determination in the mouse. Nodal flow is generated by motile node cell monocilia and requires the axonemal dynein, left-right dynein (lrd). In the absence of lrd, LR determination becomes random. The cation channel polycystin-2 is also required to establish LR asymmetry. We show that lrd localizes to a centrally located subset of node monocilia, while polycystin-2 is found in all node monocilia. Asymmetric calcium signaling appears at the left margin of the node coincident with nodal flow. These observations suggest that LR asymmetry is established by an entirely ciliary mechanism: motile, lrd-containing monocilia generate nodal flow, and nonmotile polycystin-2 containing cilia sense nodal flow initiating an asymmetric calcium signal at the left border of the node.     

Thermomechanical analysis of soft-tissue thermotherapy

Soft-tissue thermotherapy based on sub-ablative heating of collagenous tissues finds wide-spread application in medicine such as tissue welding, thermokeratoplasty, skin resurfacing, elimination of discogenic pain in the spine and treatment of joint instability. In this paper, heat-induced thermomechanical response characteristics of collagenous tissues are quantified by means of in vitro experimentation with a representative model tissue (New Zealand white rabbit patellar tendon). Three distinct heat-induced thermomechanical response regimes (defined by the rate of deformation and the variation of material properties) are identified. Arrhenius damage integral representation of collagenous tissue thermal history is shown to be adequate in establishing the master response curves for quantification of thermomechanical response for modeling purposes. The trade-off between the improved kinematical stability and compromised mechanical stability of the heated collagenous tissue is shown to be the major challenge hindering the success of subablative thermotherapies.     

The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins

Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.     

The copper transport protein Atox1 promotes neuronal survival

Atox1, a copper transport protein, was recently identified as a copper-dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.     

Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

Sensitive method to identify and characterize proteinases in situ after SDS-PAGE

Cells and body fluids contain numerous, different proteinases; to identify and characterize them are both important and difficult tasks. Especially difficult to identify and characterize are highly specific proteinases. Here, we present an extremely sensitive and quantitative method to characterize proteinases fractionated by SDS-PAGE that cleave specific rhodamine-based fluorogenic substrates. To test the sensitivity of the technique, we used trypsin as our model system. Filter paper impregnated with rhodamine-based fluorogenic substrates was placed on a gel, and bands of fluorescence originating from specific proteinases were visualized in real time. The method is very sensitive; picogram amounts of trypsin can be detected. The method should be very general, in that even proteinases whose substrates require amino acids C-terminal to the cleavage site may be identified and characterized. The results allow one to obtain not only information on the substrate specificity of a specific enzyme but also information about its molecular weight.