A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformationmediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkö rper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkö rper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubulebased motor protein is essential for normal positioning of the Spitzenkö rper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.
BACKGROUND:Measurements of free thyroxine (FT 4 ) and free triiodothyronine (FT 3 ) are important for the diagnosis and monitoring of thyroid diseases. Considerable differences among methods limit their clinical utility, however, and accurate methods are needed for various clinical specimens. We describe a direct equilibrium dialysis (ED)-liquid chromatography (LC)/ tandem mass spectrometry (MS/MS) method for FT 4 and FT 3 .
To the Editor:Vitamin D [ergocalciferol (D 2 ) and cholocalciferol (D 3 )] is 25-hydroxylated by the liver and subsequently hydroxylated by the kidney to form 1,25-vitamin D, the active form of the vitamin. 25-Hydroxyvitamin D (25-OH-vitamin D) and 1,25-dihydroxyvitamin D have important effects on calcium absorption, bone calcium balance, and renal excretion of calcium and phosphorus (1 ). Assessment of the monohydroxy form of vitamin D is important for identifying insufficient endogenous synthesis disorders that impair gastrointestinal absorption and to identify renal or hepatic abnormalities (2 ).Here
This paper describes the development and preliminary testing of a competitive surface-enhanced Raman scattering (SERS) immunoassay for calcitriol, the 1,25-dihydroxy metabolite (1,25-(OH)(2)-D(3)) of vitamin D(3). Deficiencies in 1,25-(OH)(2)-D have been linked to renal disease, while elevations are linked to hypercalcemia. Thus, there has been a sharp increase in the clinical demand for measurements of this metabolite. The work herein extends the many attributes of SERS-based sandwich immunoassays that have been exploited extensively in the detection of large biolytes (e.g., DNA, proteins, viruses, and microorganisms) into a competitive immunoassay for the low level determination of a small biolyte, 1,25-(OH)(2)-D(3) (M(w) = 416 g mol(-1)). The assay uses surface modified gold nanoparticles as SERS labels, and has a dynamic range of 10-200 pg mL(-1) and a limit of detection of 8.4 ± 1.8 pg mL(-1). These analytical performance metrics match those of tests for 1,25-(OH)(2)-D(3) that rely on radio- or enzyme-labels, while using a much smaller sample volume and eliminating the disposal of radioactive wastes. Moreover, the SERS-based data from pooled-patient sera show strong agreement with that from radioimmunoassays. The merits and potential utility of this new assay are briefly discussed.
Background: Transcriptional transactivation is a process with remarkable tolerance for sequence diversity and structural geometry. In studies of the features that constitute transactivating functions, acidity has remained one of the most common characteristics observed among native activation domains and activator peptides.
During puberty, serum steroid concentrations change dramatically. The objective of this study was to determine the adrenal steroid concentrations in children from 7 to 17 years of age. Tanner stage was determined in each child by physical examination. 11-Deoxycortisol, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone and testosterone were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Androstenedione and dehydroepiandrosterone sulfate were measured by immunoassay. The median and central 95% of the steroid concentrations were determined for age, gender, and Tanner stage. Except for 11-deoxycortisol, all of the steroids exhibited an increase in concentration after age 7-9 years in both boys and girls. 11-Deoxycortisol, which is made exclusively in the adrenal cortex, declined with age and Tanner stage. This suggests that a rise in gonadal function and decreased efficiency of 11beta-hydroxylase with age may contribute to an increase in the remaining steroids. Testosterone concentrations increased more dramatically in boys, but increases were seen with each Tanner stage in girls.
From libraries of peptides and protein fragments, several inhibitors that block pheromone response in Saccharomyces cerevisiae have been isolated previously. In many cases, the inhibitors are displayed as part of a scaffold, such as green fluorescent protein. Each of the inhibitors has a characteristic physiological strength or genetic penetrance. In this report, the roles of expression level and display scaffold on the activities of a subset of pheromoneresponse pathway inhibitors were examined. Special consideration was given to the relationship between expression levels of specific inhibitors, which may exceed 50 mM in some instances, and penetrance.
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