Synthesis of tropomyosin in myogenic cultures and in RNA-directed cell-free systems: qualitative changes in the polypeptides

The synthesis of polypeptides with the properties of alpha and beta tropomyosin was investigated in differentiating cultures of a myogenic cell line and in a wheat germ cell-free system directed by purified RNA extracted at different stages of differentiation. The polypeptides co-migrate with tropomyosin in isoelectric focusing and SDS two-dimensional gel electrophoresis and SDS-urea/SDS two-dimensional gels. Like authentic tropomyosin, these polypeptides change their mobility greatly in the presence of urea and do not become labeled with proline. The beta tropomyosin synthesized in the intact cells and in the cell-free system can be separated by isoelectric focusing into at least two components. One component (designated beta1) is present in a small amount at all developmental stages examined, and a more basic component (beta2) is specific for differentiated cultures. The synthesis of beta2 in the intact cells and the capacity of purified RNA to direct its synthesis in a cell-free system become detectable and increase greatly during the period of fusion of the mononucleated cells into multinucleated fibers. The results suggest that the beta1 and beta2 tropomyosins are coded for by different genes.     

Cooperativity of lectin binding to lymphocytes,and its relevance to mitogenic stimulation

The relationship between the binding patterns of soybean agglutinin, peanut agglutinin (both in their native (unaggregated) form and in their polymerized form), and of Phaseolus vulgaris leucoagglutinin, to neuraminidase-treated lymphocytes from different sources, and the mitogenic activity of these lectins, was studied. In all cases investigated, binding of a lectin to lymphocytes which resulted in stimulation was a positive cooperative process. Our findings support the assumption that clustering of receptors and conformational changes in membrane structure are prerequisites for mitogenic stimulation.     

A systematic review of the literature examining the diagnostic efficacy of measurement of fractionated plasma free metanephrines in the biochemical diagnosis of pheochromocytoma

BACKGROUND: Fractionated plasma metanephrine measurements are commonly used in biochemical testing in search of pheochromocytoma. METHODS: We aimed to critically appraise the diagnostic efficacy of fractionated plasma free metanephrine measurements in detecting pheochromocytoma. Nine electronic databases, meeting abstracts, and the Science Citation Index were searched and supplemented with previously unpublished data. Methodologic and reporting quality was independently assessed by two endocrinologists using a checklist developed by the Standards for Reporting of Diagnostic Studies Accuracy Group and data were independently abstracted. RESULTS: Limitations in methodologic quality were noted in all studies. In all subjects (including those with genetic predisposition): the sensitivities for detection of pheochromocytoma were 96%-100% (95% CI ranged from 82% to 100%), whereas the specificities were 85%-100% (95% CI ranged from 78% to 100%). Statistical heterogeneity was noted upon pooling positive likelihood ratios when those with predisposition to disease were included (p < 0.001). However, upon pooling the positive or negative likelihood ratios for patients with sporadic pheochromocytoma (n = 191) or those at risk for sporadic pheochromocytoma (n = 718), no statistical heterogeneity was noted (p = 0.4). For sporadic subjects, the pooled positive likelihood ratio was 5.77 (95% CI = 4.90, 6.81) and the pooled negative likelihood ratio was 0.02 (95% CI = 0.01, 0.07). CONCLUSION: Negative plasma fractionated free metanephrine measurements are effective in ruling out pheochromocytoma. However, a positive test result only moderately increases suspicion of disease, particularly when screening for sporadic pheochromocytoma.     

Neuronal assemblies: single cortical neurons are obedient members of a huge orchestra

Spontaneous cortical activity of single neurons is often either dismissed as noise, or is regarded as carrying no functional significance and hence is ignored. Our findings suggest that such concepts should be revised. We explored the coherent population activity of neuronal assemblies in primary sensory area in the absence of a sensory input. Recent advances in real-time optical imaging based on voltage-sensitive dyes (VSDI) have facilitated exploration of population activity and its intimate relationship to the activity of individual cortical neurons. It has been shown by in vivo intracellular recordings that the dye signal measures the sum of the membrane potential changes in all the neuronal elements in the imaged area, emphasizing subthreshold synaptic potentials and dendritic action potentials in neuronal arborizations originating from neurons in all cortical layers whose dendrites reach the superficial cortical layers. Thus, the VSDI has allowed us to image the rather illusive activity in neuronal dendrites that cannot be readily explored by single unit recordings. Surprisingly, we found that the amplitude of this type of ongoing subthreshold activity is of the same order of magnitude as evoked activity. We also found that this ongoing activity exhibited high synchronization over many millimeters of cortex. We then investigated the influence of ongoing activity on the evoked response, and showed that the two interact strongly. Furthermore, we found that cortical states that were previously associated only with evoked activity can actually be observed also in the absence of stimulation, for example, the cortical representation of a given orientation may appear without any visual input. This demonstration suggests that ongoing activity may also play a major role in other cortical function by providing a neuronal substrate for the dependence of sensory information processing on context, behavior, memory and other aspects of cognitive function.     

Solid-phase PCR in microwells: effects of linker length and composition on tethering, hybridization, and extension

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells are suitable for enzymatic detection of immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer. The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.     

Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy

Twenty flavonoid compounds of five different subclasses were selected, and the relationship of their structure to the inhibition of low-density lipoprotein (LDL) oxidation in vitro was investigated. The most effective inhibitors, by either copper ion or 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) induction, were flavonols and/or flavonoids with two adjacent hydroxyl groups at ring B. In the presence of the later catechol group, the contribution of the double bond and the carbonyl group at ring C was negligible. Isoflavonoids were more effective inhibitors than other flavonoid subclasses with similar structure. Substituting ring B with hydroxyl group(s) at 2' position resulted in a significantly higher inhibitory effect than by substituting ring A or ring B at other positions. The type of LDL inducer had no effect in flavonoids with catechol structure. Calculated heat of formation data (deltadeltaH(f)) revealed that the donation of a hydrogen atom from position 3 was the most likely result, followed by that of a hydroxyl from ring B. Position 3 was favored only in the presence of conjugated double bonds between ring A to ring B. This study makes it possible to assign the contribution of different functional groups among the flavonoid subclasses to in vitro inhibition of LDL oxidation.     

Development of a bioassay for detection of Wnt-binding affinities for individual frizzled receptors

Wnts are secreted lipid-modified glycoproteins that carry out various signaling functions during development and in adult tissue. Wnt signaling is mediated by frizzled receptors (Fzds) at the cell surface and can be modulated by the secreted frizzled-related proteins (SFRPs) and other molecular antagonists. Abnormal Wnt signaling has been implicated in several diseases. However, due to the complexity of the Wnt signal and the lack of knowledge pertaining to the binding properties of different Wnt ligands, no therapeutic agents that target this pathway exist. Using a novel enzyme-linked immunosorbent assay (ELISA)-based technique, we were able to determine the first measurements of binding affinity for specific Wnt interactions. This study shows that purified Wnt3a, Wnt7a, and Wnt5a have different binding specificities for Fzds and SFRPs.     

Rhomboid cleaves Star to regulate the levels of secreted Spitz

Intracellular trafficking of the precursor of Spitz (Spi), the major Drosophila EGF receptor (EGFR) ligand, is facilitated by the chaperone Star, a type II transmembrane protein. This study identifies a novel mechanism for modulating the activity of Star, thereby influencing the levels of active Spi ligand produced. We demonstrate that Star can efficiently traffic Spi even when present at sub-stoichiometric levels, and that in Drosophila S(2)R(+) cells, Spi is trafficked from the endoplasmic reticulum to the late endosome compartment, also enriched for Rhomboid, an intramembrane protease. Rhomboid, which cleaves the Spi precursor, is now shown to also cleave Star within its transmembrane domain both in cell culture and in flies, expanding the repertoire of known Rhomboid substrates to include both type I and type II transmembrane proteins. Cleavage of Star restricts the amount of Spi that is trafficked, and may explain the exceptional dosage sensitivity of the Star locus in flies.