Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity

A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells. The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II₂, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons. The location of GP II₂on the cell surface was confirmed by the fact that it could be labeled metabolically with, D-(³H) glucosamine and externally through the nonpenetrating periodate-NaB³H₄ system. GP II₂could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II₂could be an integral protein. Analysis of the carbohydrate composition of GP II₂ revealed galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O- and N-linked to the polypeptide chain, most of them being O-linked. Finally, GP II₂has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.     

Immunological properties of gap junction protein from mouse liver

Hepatic gap junctions were purified as plaques from BALB/c mice and separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Antisera were raised in rabbits and rats against gap junction plaques as well as protein bands of the following apparent molecular weights: 44K to 49K ("dimer" proteins), 26K, and 21K. Using an enzyme immunoassay, we found that the reactivities of the different antisera towards gap junction plaques decreased in the following order: anti-plaque antisera, anti-26K antisera, anti-"dimer" protein antisera, and anti-21K antisera. The gap junction protein bands separated by SDS-polyacrylamide gel electrophoresis were transferred by blotting onto nitrocellulose paper and the immunological cross-reactivities were compared: the anti-26K antisera reated with the dimer protein bands and the 26K band but did not cross-react with the 21K protein band. The rabbit anti-21K antiserum reacted weakly with the 21K protein. The missing immunological cross-reaction of the 26K and the 21K protein band can be most easily explained if both proteins were independent of each other. No inhibition of metabolic cooperation between fibroblastoid mouse 3T6 cells was observed in the presence of Fab fragments prepared from rabbit antiplaque antiserum or from rabbit anti 26K antiserum. When the total proteins of plasma membranes from mouse liver were separated by SDS-polyacrylamide electrophoresis, only the 26K protein reacted with rabbit anti 26K antiserum. This result opens the possibility for direct quantitation of gap junction protein in tissues and cell fractions.     

Circular DNA in human and boar spermatozoa

Circular DNA molecules were isolated from human and boar whole spermatozoa or spermatozoal nuclei and measured for size by electron microscopy. The DNA molecules derived from both mammals were heterogeneous in size ranging from 0.07 to 17 μm; nearly 75% of the molecules were ?0.5 μm in length. The mean lengths were 1.0 μm and 1.5 μm for circular DNAs isolated from human and boar spermatozoa, respectively. The origin and function of these molecules remains unknown.     

Calcium-Activated ATPases in Presynaptic Nerve Endings

We studied the properties of calcium-activated ATPases present in preparations of isolated presynaptic nerve ending (synaptosome) and its subfractions from mouse brain. ATPase activity in the preparation was stimulated by Ca2+ and by Mg2+, but not by Na+ and K+, when each was added alone. The substrate specificities were found to be similar. The ATPases hydrolyzed only the high-energy phosphate bond and similar activity was exhibited for all nucleoside triphosphates tested (ATP, CTP, GTP, UTP). Moreover, the enzymes were insensitive to mitochondrial markers and to ouabain, but were inhibited by La3+. La3+ produced uncompetitive inhibition of Ca2+-ATPase in intact synaptosomes. Inhibition by La3+ was greatly increased after lysis of the synaptosomes, suggesting that the active sites of the enzymes may be on the cytosolic face of the membranes. The Ca2+-ATPase activity in synaptosomes was increased by increasing concentrations of external K+, suggesting that Ca2+ influx may be involved The Ca2+-ATPase in synaptosomal plasma membranes and synaptic vesicles had higher specific activities than those of intact synaptosomes and were activated, both in the presence and the absence of Mg2+, by Ca2+ concentrations approximating the intracellular level (10(-7) M). It is concluded that the nonmitochondrial synaptosomal Ca2+-ATPase may play an important role in the regulation of intracellular Ca2+.     

ACTH1–24 Releases a Protein from Synaptosomal Plasma Membranes

Abstract: Brain membranes contain several protein kinases, all of which appear to play a role in the regulation of neuronal functioning. These membranes also contain numerous (phospho) proteins. It has been proposed that the degree of phosphorylation of some of these proteins may affect neuronal membrane properties. In a series of previous reports we showed that ACTH_1-24 inhibits the endogenous phosphorylation of several synaptosomal plasmamembrane (SPM) proteins including the B-50 protein. Although we have speculated that the degree of phosphorylation of B-50 may be important in regulating the turnover of membrane (poly)-phosphoinositides, the exact nature of the interaction between ACTH_1-24 and B-50/B-50 protein kinase is unknown. The purpose of the present study was to determine whether treatment of SPM with ACTH_1-24 will lead to a specific release of proteins from SPM. We found that ACTH_1-24 specifically releases a 41,000 M_r protein from rat brain SPM. Although we are not certain about the biological significance of the release of this polypeptide, it is of sufficient interest for further research in view of the lack of success of finding binding of labeled ACTH to brain membranes.     

Terrestrial predator alarm vocalizations are a valid monitor of stress in captive brown capuchins (Cebus apella)

The vocal behavior of captive animals is increasingly exploited as an index of well‐being. Here we show that the terrestrial predator alarm (TPA) vocalization, a robust and acoustically distinctive anti‐predation vocal response present in many mammal and bird species, offers useful information on the relative well‐being and stress levels of captive animals. In a 16‐week experiment evaluating the effects of varying levels of physical environmental enrichment (control < toys < foraging box < foraging box and toys) in the cages of eight singly housed adult male brown capuchins, we quantified the 1) emission rate of TPAs, 2) proportions of normal and abnormal behavior sample intervals, and 3) fecal and plasma cortisol levels. Variation in TPA emission across the experimental conditions was significant. We found significant reductions in the mean TPA production rate by the group in the enriched (toys, foraging box, and foraging box and toys) compared to the control condition; pre‐ and post‐experimental conditions, however, did not differ from the control condition. Mean TPA production by the group was also significantly positively correlated to mean group levels of fecal cortisol and proportion of abnormal behavior sample intervals, and significantly negatively correlated to the average proportion of normal behavior sample intervals in the group. Based on group means, plasma cortisol levels were positively, but not significantly, related to increasing TPA rate. At the level of the responses of an individual subject, however, the covariation between the vocal and non‐vocal behavioral measures and the cortisol assays seldom attained significance. Nevertheless, the direction of the relationships among these parameters within individual subjects typically mirrored those correlations based on group means. At both the group mean and individual levels, our results are consistent with the interpretation that in conditions of low environmental enrichment the study subjects were more stressed, and therefore more reactive to the presence of a threatening terrestrial stimulus (human observer), than when in more enriched conditions. We suggest that protocols to evaluate the effectiveness of enrichment for captive species other than brown capuchins could also profitably exploit TPAs as a first‐line monitor or as corroboratory evidence of current well‐being. Zoo Biol 18:295–312, 1999. © 1999 Wiley‐Liss, Inc.     

Chlortetracycline analysis of boar spermatozoa after incubation,flow cytometric sorting,cooling, or cryopreservation

In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC-pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR-14 and PI. Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of spermatozoa allocated to the F (uncapacitated), B (capacitated), or AR (acrosome-reacted) CTC fluorescent staining categories. The mean percentages of acrosome-intact and acrosome-reacted cells were 88.4 and 6.8 or 0.8 and 96.5 in semen treated with 0 or 100 μg/ml lysophosphatidylchloine (LPC), respectively (P < 0.001). Most spermatozoa were also in the AR CTC-stained category after treatment with LPC compared with a small proportion in the controls. Using flow cytometry to examine sperm suspensions stained with CTC, a gated population of spermatozoa with low fluorescence (population 1) comprised predominantly F-pattern cells (F-pattern: population 1 vs. population 2, 80.5 vs. 14.4%; P < 0.001), whereas population 2 (high fluorescence) comprised mainly B-pattern cells (B-pattern: population 1 vs. population 2, 8.5 vs. 62.3%; P < 0.001). Incubation (38°C, 4 hr), flow sorting, cooling (to 15 or 5°C) and freezing reduced the proportion of F-pattern and live spermatozoa, and increased the proportion of B-, AR-pattern, and dead spermatozoa, in comparison with fresh semen. There were more membrane changes in spermatozoa cooled to 5°C (30.4, 48.5, 21.1%) than in those cooled to 15°C (56.1, 32.6, 11.5% F-, B-, and AR-pattern spermatozoa, respectively). Mol. Reprod. Dev. 46:408–418, 1997. © 1997 Wiley-Liss, Inc.     

Immobilization of biomolecular anthocyanin natural sensor into plasma-cured polyethylene terephthalate fibers from recycled plastic waste for determination of ammonia

Polyester textiles have been applied in numerous industrial applications. Polyester fibers are characterized with being excellent insulators to electricity, having excellent flexural and impact strength, ease of manufacture, low-cost, as well as having resistance to moisture and chemicals. However, polyester fibers cannot be stained due to the absence of active dyeing sites on the surface of the fibrous structure. Thus, polyester cannot be dyed after it has been extruded. Herein, we report the development of novel-colored polyester fabrics using plasma-assisted dyeing and anthocyanin natural probe for determination of ammonia that may cause severe harmful effects to human organs and even death. Anthocyanin was extracted from red cabbage and characterized. The water-soluble anthocyanin was fastened to polyester fibers by mordant (potash alum) to generate anthocyanin–mordant coordinative complex nanoparticles. Polyester can be treated with thin layer of anthocyanin probe after activation with plasma. The results showed excellent colorfastness, ultraviolet blocking, and antibacterial performance of the anthocyanin-dyed polyester (APET) fibers. The APET fibers showed great potential for developing a portable colorimetric device for an on-site detection of ammonia. APET displayed a detection limit of aqueous ammonia in the range of 25–200 ppb, displaying a change in color from purple (542 nm) to white (387 nm).     

Predicting the gender of subadult Komodo dragons (Varanus komodoensis) using two-dimensional ultrasound imaging and plasma testosterone concentration

Six yearling Komodo dragons (Varanus komodoensis) underwent ultrasound examinations every three months over a nine-month period to assess the value of two-dimensional ultrasound imaging technology for the purpose of determining gender in this highly monomorphic species. Beginning at an age of 28 months, ovarian structures could be imaged, allowing a prediction of gender based on the presence or absence of ovarian follicles. Simultaneously, plasma testosterone concentration was measured monthly on each individual during the study period. Beginning at an age of 24 months, putative males had consistently elevated plasma testosterone values that were ∼40–60 times greater than those of putative females. In a blind comparison, predictions of gender based on plasma testosterone values matched the prediction of sex based on ultrasound imaging for all six individuals. The results indicate that measurements of circulating testosterone and two-dimensional ultrasound imaging together appear useful in the noninvasive determination of gender in juvenile V. komodoensis at just over two years of age. © 1996 Wiley-Liss, Inc.