Sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal

A multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM) principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin was used in this study, M _r6000 Da) was correlated with the shift of resonant frequency of QCM system before and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity, and is sensitive to approx. 1 ng insulin ml^–1 with a linear response for insulin from 10 g ml^–1 to 10 ng ml^–1 in standard solutions. The technique may also be applied for the detection of other small biomolecules.     

Direct interaction of soluble human recombinant tau protein with Abeta 1-42 results in tau aggregation and hyperphosphorylation by tau protein kinase II

We report here that aggregated beta-amyloid (Abeta) 1-42 promotes tau aggregation in vitro in a dose-dependent manner. When Abeta-mediated aggregated tau was used as a substrate for tau protein kinase II (TPK II), an 8-fold increase in the rate of TPK II-mediated tau phosphorylation was observed. The extent of TPK II-dependent tau phosphorylation increased as a function of time and Abeta 1-42 concentration, and hyperphosphorylated tau was found to be decorated with an Alzheimer's disease-related phosphoepitope (P-Thr-231). In HEK 293 cells co-expressing CT-100 amyloid precursor protein and tau, the release of Abeta 1-42 from these cells was impaired. Taken together, these in vitro results suggest that Abeta 1-42 promotes both tau aggregation and hyperphosphorylation.     

Conformational control through translocational regulation: a new view of secretory and membrane protein folding

We suggest a new view of secretory and membrane protein folding that emphasizes the role of pathways of biogenesis in generating functional and conformational heterogeneity. In this view, heterogeneity results from action of accessory factors either directly binding specific sequences of the nascent chain, or indirectly, changing the environment in which a particular domain is synthesized. Entrained by signaling pathways, these variables create a combinatorial set of necessary-but-not-sufficient conditions that enhance synthesis and folding of particular alternate, functional, conformational forms. We therefore propose that protein conformation is productively regulated by the cell during translocation across the endoplasmic reticulum (ER), a concept that may account for currently poorly understood aspects of physiological function, natural selection, and disease pathogenesis.     

Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.     

Diagnostic DNA testing for X-linked ocular albinism (OA1) with a hierarchical mutation screening protocol

Albinism is a group of inherited conditions in which affected individuals have less than normal pigment in the eyes, skin, and hair compared to others of the same race and ethnic background. The prevalence of all types of albinism in the United States is estimated at 1 in 20,000, based on poor epidemiological data. X-linked Nettleship-Falls ocular albinism (XLOA, OA1) affects approximately 1/150,000 males in the population. XLOA effects reduce visual acuity and nystagmus, result in a mild skin and hair phenotype, and occur mostly in XY males. Female carriers of XLOA have normal visual acuity, but often show iris punctate transillumination and a classic pattern of mosaic retinal pigmentation, coarse and grainy in the macula and becoming increasingly reticular into the periphery of the retinal pigment epithelium. Studies of OA1 have shown linkage of a single gene to markers at Xp22.3-p22.2. About 48% of the reported mutations in the OA1 gene are intragenic deletions and about 43% are point mutations. We present a hierarchical strategy for mutation screening for diagnostic testing for OA1 that comprises two tiers: first, multiplex PCR to detect intragenic deletions in the OA1 gene with denaturing high-performance liquid chromatography (dHPLC), and, second, heteroduplex analysis with dHPLC to scan for mutations, with subsequent sequencing of variants to confirm putative mutations in the OA1 gene. Prenatal diagnosis can be provided for families when the mutation has been firmly identified. We have validated this procedure with positive controls that were identified in patients by Southern blot, single-stranded conformation polymorphism (SSCP), and sequencing. In this hierarchical strategy, these procedures have an analytical sensitivity of > 99%.     

Androgen receptor expression and DNA content of paraffin-embedded archival human prostate tumors

BACKGROUND: Androgen receptors (AR) are expressed in human prostate cells and immunohistochemistry has been used for qualitative analysis of AR expression in prostate tumor cells. Quantitative and multiparametric analysis of receptor expression could be of diagnostic and prognostic value in the management of patients on antiandrogen therapy. Multiparametric flow cytometric methods have been developed for analysis of hormone receptor expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded human solid tumors. The present study was undertaken for analysis of AR expression and DNA content in archival human prostate tumors. METHODS: AR expression and DNA content were measured in nuclei isolated by enzyme digestion from thick sections cut from 51 paraffin-embedded human prostate tumors. AR expression in different subpopulations was studied by gated analysis. The relationship among AR activity, DNA content, and histopathological grade was analyzed. RESULTS: Distinct aneuploid populations were observed in 23% of tumors examined. AR activity was observed in all the specimens and the percentage of AR- positive nuclei in the 48 samples analyzed was <10% (n = 4), 11-50% (n = 39), and >51% (n = 5). Tumor subpopulations with aneuploid DNA content had higher AR expression (percent AR-positive cells and mean log fluorescence) than the diploid subpopulations. No strong correlation was seen between AR expression and histopathological grade of the tumors. CONCLUSIONS: Flow cytometric analysis of archival prostate tumor can be used for rapid determination of aneuploid DNA content and AR expression in subpopulations of nuclei isolated from formalin-fixed/paraffin-embedded prostate tumor blocks.     

Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II

The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents. We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rho-associated kinase (ROK). Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60(c-src), p(190) RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of Galpha13, carboxyl terminus peptide, and ADP ribosylation factor (ARF) antibody. Ang II-induced increase in p(190) RhoGAP tyrosine phosphorylation was attenuated by genistein. Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genistein. The results suggest an important role for Galpha13 and pp60(c-src) in the intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.