Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

THE DIRECT MEASUREMENT OF THE AXOPLASMIC TRANSPORT OF INDIVIDUAL RNA SPECIES: TRANSFER BUT NOT RIBOSOMAL RNA IS TRANSPORTED

Analysis of chick retinal and tectal RNA revealed that in addition to the major cytoplasmic RNAs (rRNA and tRNA), a number of the small mol wt nuclear RNAs (snRNAs) can also be detected. Subfractionation data indicated that one of these molecules, DD′, is of at least 95% nuclear location within the retina. Thus, very little, if any, of the retinal DD′ is available for axoplasmic transport from the retina into the optic nerve and tectum. Following intraocular injection of [³H]uridine, considerable incorporation of isotope into DD′ was observed within the optic tectum after 4, 8 and 16 days. This result indicates the presence of considerable local (i.e. tectal) synthesis. The specific activities of 29S, 18S and 5S rRNA and 4s tRNA relative to that of DD′ were measured in the optic tectum 8 and 16 days after the intraocular introduction of [³H]uridine. The same measurements were also made in intracranially injected animals. While the 29S/DD′, 18S/DD′ and 5S/DD′ specific activity ratios obtained were independent of the injection route, the 4S/DD′ ratio obtained from intraocularly injected animals was significantly greater (at least 2-fold) than that obtained from intracranially injected animals. Similar analysis was also performed with the optic nerve complex at 16 days post-injection with identical results. These results demonstrate that tRNA, but not rRNA, is transported from the retina into the optic nerve and tectum in the 2-day-old chicken.     

Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.     

Effects of Enucleation on High-Affinity Binding Sites in Chick Optic Tecta

Abstract: The effect of unilateral eye extirpation on the development of the chick optic tectum has been studied in both the embryo and the newly hatched chick. Although the prevention of normal afferentation of the embryonic tectum retarded its growth, there appeared to be a significant increase of muscarinic acetylcholine binding site in the noninnervated tectum. This phenomenon was repeated also in the posthatch denervated system wherein the functioning optic nerve is severed. A significant increase in the number of binding sites as well as reduced dissociation constant of the interactions of this receptor with [³H]quinuclindinyl benzilate was found in the deafferented optic tectum. This may suggest the presence of a denervation-supersensitivity-like modulation. Similar increases were not detected with other binding sites studied in either the noninnervated embryonic or deafferented posthatch optic lobes. The possibility that acetylcholine is a primary neurotransmitter of the optic system is discussed.     

Programmatic Cost Evaluation of Nontargeted Opt-Out Rapid HIV Screening in the Emergency Department

Background

The Centers for Disease Control and Prevention recommends nontargeted opt-out HIV screening in healthcare settings. Cost effectiveness is critical when considering potential screening methods. Our goal was to compare programmatic costs of nontargeted opt-out rapid HIV screening with physician-directed diagnostic rapid HIV testing in an urban emergency department (ED) as part of the Denver ED HIV Opt-Out Trial.

Methods

This was a prospective cohort study nested in a larger quasi-experiment. Over 16 months, nontargeted rapid HIV screening (intervention) and diagnostic rapid HIV testing (control) were alternated in 4-month time blocks. During the intervention phase, patients were offered HIV testing using an opt-out approach during registration; during the control phase, physicians used a diagnostic approach to offer HIV testing to patients. Each method was fully integrated into ED operations. Direct program costs were determined using the perspective of the ED. Time-motion methodology was used to estimate personnel activity costs. Costs per patient newly-diagnosed with HIV infection by intervention phase, and incremental cost effectiveness ratios were calculated.

Results

During the intervention phase, 28,043 eligible patients were included, 6,933 (25%) completed testing, and 15 (0.2%, 95% CI: 0.1%–0.4%) were newly-diagnosed with HIV infection. During the control phase, 29,925 eligible patients were included, 243 (0.8%) completed testing, and 4 (1.7%, 95% CI: 0.4%–4.2%) were newly-diagnosed with HIV infection. Total annualized costs for nontargeted screening were $148,997, whereas total annualized costs for diagnostic HIV testing were $31,355. The average costs per HIV diagnosis were $9,932 and $7,839, respectively. Nontargeted HIV screening identified 11 more HIV infections at an incremental cost of $10,693 per additional infection.

Conclusions

Compared to diagnostic testing, nontargeted HIV screening was more costly but identified more HIV infections. More effective and less costly testing strategies may be required to improve the identification of patients with undiagnosed HIV infection in the ED.     

An integrated approach to the ligand binding specificity of Neisseria meningitidis M1 alanine aminopeptidase by fluorogenic substrate profiling,inhibitory studies and molecular modeling

Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria.     

The relationship between airborne pollen and fungal spore concentrations and seasonal pollen allergy symptoms in Cracow in 1997–1999

The investigation of airborne pollen and fungalspore concentrations was carried out in Cracowbetween 1997–1999. For this study thevolumetric method has been employed (Burkard).At the same time the clinical diagnosis ofpollen allergy in 40 patients was obtained onthe basis of an interview, positive skin pricktests with pollen extracts and increasedspecific IgE level. An increase in seasonalallergy symptoms in all patients occurred fromthe middle of May to the middle of August.Eighty eight percent of the patients (35 out of40) were sensitive to Poaceae pollen and about50% of them were additionally sensitive totree and herb pollen excluding grasses. Forpatients with additional allergy to tree pollenthe seasonal symptoms started at the end ofMarch (Betula) while for patients withadditional allergy to herb pollen it wasextended to the middle of September (Artemisia).Five people out of 40 revealed positive skinreactions to Alternaria spores and anincrease in specific IgE level. Positive skinreaction to Cladosporium spores with noincrease in specific IgE level occurred in 2patients. The increase in seasonal allergysymptoms in people sensitive to Alternariaspores noted in July and August could becaused not only by these spores but also byPoaceae pollen.     

Automated Bayesian model development for frequency detection in biological time series

Background

The ability to perform quantitative studies using isotope tracers and metabolic flux analysis (MFA) is critical for detecting pathway bottlenecks and elucidating network regulation in biological systems, especially those that have been engineered to alter their native metabolic capacities. Mathematically, MFA models are traditionally formulated using separate state variables for reaction fluxes and isotopomer abundances. Analysis of isotope labeling experiments using this set of variables results in a non-convex optimization problem that suffers from both implementation complexity and convergence problems.

Results

This article addresses the mathematical and computational formulation of ¹³C MFA models using a new set of variables referred to as fluxomers. These composite variables combine both fluxes and isotopomer abundances, which results in a simply-posed formulation and an improved error model that is insensitive to isotopomer measurement normalization. A powerful fluxomer iterative algorithm (FIA) is developed and applied to solve the MFA optimization problem. For moderate-sized networks, the algorithm is shown to outperform the commonly used 13CFLUX cumomer-based algorithm and the more recently introduced OpenFLUX software that relies upon an elementary metabolite unit (EMU) network decomposition, both in terms of convergence time and output variability.

Conclusions

Substantial improvements in convergence time and statistical quality of results can be achieved by applying fluxomer variables and the FIA algorithm to compute best-fit solutions to MFA models. We expect that the fluxomer formulation will provide a more suitable basis for future algorithms that analyze very large scale networks and design optimal isotope labeling experiments.