Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Oriented thick and thin filaments in amoeba proteus

Actin and myosin filaments as a foundation of contractile systems are well established from ameba to man (3). Wolpert et al. (19) isolated by differential centrifugation from Amoeba proteus a motile fraction composed of filaments which moved upon the addition of ATP. Actin filaments are found in amebas (1, 12, 13) which react with vertebrate heavy meromyosin (HMM), forming arrowhead complexes as vertebrate actin (3, 9), and are prominent within the ectoplasmic tube where some of them are attached to the plasmalemma (1, 12). Thick and thin filaments possessing the morphological characteristics of myosin and actin have been obtained from isolated ameba cytoplasm (18, 19). In addition, there are filaments exhibiting ATPase activity in amebas which react with actin (12, 16, 17). However, giant ameba (Chaos-proteus) shapes are difficult to preserve, and the excellent contributions referred to above are limited by visible distortions occurring in the amebas (rounding up, pseudopods disappearing, and cellular organelles swelling) upon fixation. Achievement of normal ameboid shape in recent glycerination work (15) led us to attempt other electron microscope fixation techniques, resulting in a surprising preservation of A. proteus with a unique orientation of thick and thin filaments in the ectoplasmic region.     

Degradation of Methylmercury by Bacteria Isolated from Environmental Samples

A total of 207 bacterial cultures, isolated from environmental samples, was screened for ability to degrade methylmercury. Of these, 30 were found positive for aerobic demethylation. Twenty-two of these were shown to be facultative anaerobes and 21 of these degraded methylmercury anaerobically. All positive species volatilized methylmercury aerobically, and methane was produced as a degradation product. Although methylmercury degradation was complete in most cases, material balances indicated some of the inorganic mercury formed was not volatilized and is presumed bound to the cells. All positive isolates were tolerant to at least 0.5 mug of methylmercury per ml, and the extent of volatilization of mercury increased with concentration to the threshold value. The results indicate that demethylating species are prevalent in the environment and may be important in suppressing the methylmercury content of sediments.     

Colicin synthesis and cell death.

Colicin E1 is a small plasmid, containing the cea gene for colicin, the most prominent product of the plasmid. Colicin is a 56-kilodalton bacteriocin which is especially toxic to Escherichia coli cells that do not contain the plasmid. Under normal growth conditions very low levels of the plasmid are produced as a result of cea gene repression by the host LexA protein. Conditions that lower the concentration of LexA protein result in elevated levels of colicin synthesis. The LexA protein concentration can be lowered by exposing the cells to DNA-damaging reagents such as UV light or mitomycin C. This is because DNA damage signals the host SOS response; the response leads to activation of the RecA protease which degrades the LexA protein. DNA-damaging reagents result in very high levels of colicin synthesis and subsequent death of plasmid-bearing cells. Elevated levels of colicin are also produced in mutants of E. coli that are deficient in LexA protein. We found that comparably high levels of colicin can be produced in such mutants in the absence of cell death. In lexA strains carrying a defective LexA repressor, colicin synthesis shows a strong temperature dependence. Ten to twenty times more colicin is synthesized at 42 degrees C. This sharp dependence of synthesis on temperature suggests that there are factors other than the LexA protein which regulate colicin synthesis.     

Dynamic properties of polyelectrolyte calcium membranes

Summary Shashoua observed spontaneous oscillations in a polyelectrolyte membrane formed by interfacial precipitates of polyacid and polybase. We have here undertaken experimental and theoretical studies of polyglutamic acid-Ca⁺⁺ membrane in order to clarify the processes involved in this dynamic behavior. We find a region of distinct hysteresis in the voltage current curve for this system. A sharp transition from a state of low membrane resistance to one of high resistance occurs at a current density different from that of inverse transition.This membrane system is modeled as a two layer structure: a negatively charged layer made of ionized polyelectrolyte in series with a neutral region in which the polymeric ionic sites are masked by calcium ion. This structure results in a difference in the transference number for the mobile ions, causing salt accumulation at the interfacial region during a current flow in the to direction. This altered salt concentration induces a change of polymeric conformation, which in turn affects the membrane permeability and the rate of accumulation. Based upon nonequilibrium thermodynamic flow equations, and a two-state representation of membrane macromolecular conformation, this model displays a region of hysteresis in the current range of experimental observations.     

The cholinesterase inhibitor, phenserine, improves Morris water maze performance of scopolamine-treated rats

Male Fischer-344 rats (n = 38) at 5 months old were tested in a Morris water maze to determine if treatment with the cholinesterase inhibitor, phenserine (PHEN), would overcome a learning impairment induced by scopolamine (SCOP), a muscarinic cholinergic receptor antagonist. Each rat was randomly assigned to one of five groups to receive two intraperitoneal injections 60 and 30 min, prior to testing, respectively, as follows: (1) saline-saline (SAL); (2) saline-1.0 mg/kg (SCOP); (3) 2 mg/kg PHEN- SCOP (PHEN2); (4) 4 mg/kg PHEN-SCOP (PHEN4); and (5) 1 mg/kg PHEN-SAL (PHEN1). Maze testing occurred across 5 days with 4 days of acquisition trials (4 trials per day) and a fifth day consisting of a single 120 sec probe trial. PHEN1 and SAL were combined into one control (CON) group for purposes of statistical analysis for both acquisition and probe trials as comparison of the two groups revealed that they did not significantly differ on any measure. SCOP-treated rats were significantly impaired compared to CON in learning the location of the submerged platform as measured by latency to locate the platform and the distance traversed to find the platform across days of testing. The PHEN4 group had significantly lower latencies and traveled a shorter distance to reach the submerged platform when compared to SCOP on the fourth day of trials while the PHEN2 group traveled more directly to the submerged platform but did not have shorter latencies than the SCOP group. For probe trials, CON rats swam closer to the target area (a measure of proximity to the removed platform) than did all other groups, and the PHEN4 group swam in an area more proximate to the target area than did the SCOP-treated group. These findings demonstrate the ability of this drug to improve learning when cholinergic function has been impaired in a spatial memory task.     

Interleukin (IL)-1, IL-6, and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives

Inflammation of the female reproductive tract increases susceptibility to HIV-1 and other viral infections and, thus, it becomes a serious liability for vaginal products. Excessive release of proinflammatory cytokines may alter the mucosal balance between tissue destruction and repair and be linked to enhanced penetration and replication of viral pathogens upon chemical insult. The present study evaluates four surface-active microbicide candidates, nonoxynol-9 (N-9), benzalkonium chloride (BZK), sodium dodecyl sulfate, and sodium monolaurate for their activity against human sperm and HIV, and their capacity to induce an inflammatory response on human vaginal epithelial cells and by the rabbit vaginal mucosa. Spermicidal and virucidal evaluations ranked N-9 as the most potent compound but were unable to predict the impact of the compounds on vaginal cell viability. Interleukin (IL)-1 release in vitro reflected their cytotoxicity profiles more accurately. Furthermore, IL-1 concentrations in vaginal washings correlated with cumulative mucosal irritation scores after single and multiple applications (P < 0.01), showing BZK as the most damaging agent for the vaginal mucosa. BZK induced rapid cell death, IL-1 release, and IL-6 secretion. The other compounds required either more prolonged or repeated contact with the vaginal epithelium to induce a significant inflammatory reaction. Increased IL-8 levels after multiple applications in vivo identified compounds with the highest cumulative mucosal toxicity (P < 0.01). In conclusion, IL-1, IL-6, and IL-8 in the vaginal secretions are sensitive indicators of compound-induced mucosal toxicity. The described evaluation system is a valuable tool in identifying novel vaginal contraceptive microbicides, selecting out candidates that may enhance, rather than decrease, HIV transmission.     

Molecular analysis of telomere fusions in Arabidopsis: multiple pathways for chromosome end-joining

End-to-end fusion of critically shortened telomeres in higher eucaryotes is presumed to be mediated by nonhomologous end-joining (NHEJ). Here we describe two PCR-based methods to monitor telomere length and examine the fate of dysfunctional telomeres in Arabidopsis lacking the catalytic subunit of telomerase (TERT) and the DNA repair proteins Ku70 and Mre11. Primer extension telomere repeat amplification relies on the presence of an intact G-overhang, and thus measures functional telomere length. The minimum functional telomere length detected was 300-400 bp. PCR amplification and sequence analysis of chromosome fusion junctions revealed exonucleolytic digestion of dysfunctional ends prior to fusion. In ku70 tert mutants, there was a greater incidence of microhomology at the fusion junction than in tert mutants. In triple ku70 tert mre11 mutants, chromosome fusions were still detected, but microhomology at the junction was no longer favored. These data indicate that both Ku70 and Mre11 contribute to fusion of critically shortened telomeres in higher eucaryotes. Furthermore, Arabidopsis processes critically shortened telomeres as double-strand breaks, using a variety of end-joining pathways.     

Phylogenetic analyses of the rbcL sequences from haptophytes and heterokont algae suggest their chloroplasts are unrelated

Using the large subunit of RuBisCo (rbcL) sequences from cyanobacteria, proteobacteria, and diverse groups of algae and green plants, we evaluated the plastid relationship between haptophytes and heterokont algae. The rbcL sequences were determined from three taxa of heterokont algae (Bumilleriopsis filiformis, Pelagomonas calceolata, and Pseudopedinella elastica) and added to 25 published sequences to obtain a data set comprising 1,434 unambiguously aligned sites (approximately 98% of the total rbcL gene). Higher levels of mutational saturation in third codon positions were observed by plotting the pairwise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In accordance with this finding phylogeny reconstructions were completed by omitting third codon positions, thus using 956 bp in weighted-parsimony and maximum-likelihood analyses. The midpoint-rooted phylogenies showed two major clusters, one containing cyanobacteria, glaucocystophytes, a phototrophic euglenoid, chlorophytes, and embryophytes (the green lineage), the other containing proteobacteria, haptophytes, red algae, a cryptophyte, and heterokont algae (the non-green lineage). In the nongreen lineage, the haptophytes formed a sister group to the clade containing heterokont algae, red algae, and the cryptophyte Guillardia theta. This branching pattern was well supported in terms of bootstrap values in weighted- parsimony and maximum-likelihood analyses (100% and 92%, respectively). However, the phylogenetic relationship among red algae, heterokonts, and a cryptophyte taxon was not especially well resolved. A four- cluster analysis was performed to further explore the statistical significance of the relationship between proteobacteria, red algae (including and excluding Guillardia theta), haptophytes, and heterokont algae. This test strongly favored the hypothesis that the heterokonts and red algae are more closely related to each other than either is to proteobacteria or haptophytes. Hence, this molecular study based on a plastid-encoded gene provides additional evidence for a distant relationship between haptophytes and the heterokont algae. It suggests an evolutionary scenario in which the ancestor of the haptophyte lineage engulfed a phototrophic eukaryote and, more recently, the heterokont lineage became phototrophic by engulfing a red alga.