In vitro differentiation of rabbit blastocyst cells

Summary Six- and seven-day post-coitus (p.c.) rabbit embryos have been cultured in an attempt to establish a trophectodermal cell line. Results indicate that cells with epithelial characteristics (i.e. positive staining for cytokeratin) will survive in culture until Passage 3. At that time a fibroblastlike cell becomes predominant. In addition, we have found that the presence of the inner cell mass is required for embryo explants often results in the development of cells that spontaneously contract. These cells stain positively for myosin, which indicates that they may be precardiac cells. Maximum diastolic potential was −59±1.2 mV and the threshold potential was −53±2.3 mV. Spontaneously contracting cells did not respond to atropine, acetylcholine, epinephrine, isoproterenol, or propranolol. Action potential seems to be a result of an inward calcium current, because the beating rate is decreased in a dose-related manner with the calcium channel blocker verapamil, whereas the voltage-sensitive sodium channel blocker tetrodotoxin was without effect. This work was supported by grants HD21302, HD07069, DK31091, and HL37320 from the National Institutes of Health, Bethesda, MD, with additional support from a University of Alabama at Birmingham Cardviovascular Research and Training Center Award.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

A pyrenoid-based carbon-concentrating mechanism is present in terrestrial bryophytes of the class Anthocerotae

It has been widely accepted that carbon assimilation in bryophytes is exclusively based on the conventional C₃ photosynthetic pathway. The occurrence of biochemical CO₂-concentrating mechanisms (C₄ or Crassulacean acid metabolism), which have developed in plants in the last 20–100 million years, has been discounted for bryophytes from studies of the carbon isotope composition (¹³C) of organic material. In contrast cyanobacteria and many algae show active accumulation of dissolved inorganic carbon via biophysical CO₂-concentrating mechanisms which are also found in the photobiont partners in certain lichens. The presence of a pyrenoid, a granular particle within the chloroplast, has been linked with CO₂-concentrating mechanism activity in green algae and lichens and we now show that such a mechanism is categorically associated with the occurrence of a pyrenoid in bryophytes belonging to the class of Anthocerotae. These observations have significant evolutionary implications for the development of terrestrial photosynthesis during the colonisation of the land, raising the intriguing question of why the pyrenoid-based CO₂-concentrating mechanism did not persist in the terrestrial environment.Abbreviations and Symbols CCM carbon-concentrating mechanism - DIG dissolved inorganic carbon (CO₂+HCO ₃ ^- +CO ₂ ^- ) - DW dry weight - K_0.5 external concentration of CO₂ at which half-maximal rates of CO₂ assimilation are reached - Rubisco ribulose-l,5-bisphosphate carboxylase-oxygenase - carbon isotope discrimination (%) - ¹³C carbon isotope ratio (%) This work was supported by the Natural Environment Research Council (GR3/8813) and the Leverhulme Trust. We thank Prof. A. Roy Perry (National Museum of Wales, Cardiff), Dr. B. Coppins and Mr. D. Long (Royal Botanic Garden Edinburgh) for access to herbarium specimens and Mr. M. Fletcher for providing living bryophytes.     

Age-Dependent Impairment of Mitochondrial Function in Primate Brain

Abstract: It has been hypothesized that some of the functional impairments associated with aging are the result of increasing oxidative damage to mitochondrial DNA that produces defects in oxidative phosphorylation. To test this hypothesis, we examined the enzymes that catalyze oxidative phosphorylation in crude mitochondrial preparations from frontoparietal cortex of 20 rhesus monkeys (5-34 years old). Samples were assayed for complex I, complex II-III, complex IV, complex V, and citrate synthase activities. When enzyme activities were corrected for citrate synthase activities (to account for variable degrees of mitochondrial enrichment), linear regression analysis demonstrated a significant negative correlation of the activities of complex I (p < 0.002) and complex IV (p < 0.03) with age but no significant change in complex II-III or complex V activities. Relative to animals 6.9 ± 0.9 years old (n = 7), the citrate synthase-corrected activity of complex I was reduced by 17% in animals 22.5 ± 0.9 years old (n = 6) (p < 0.05) and by 22% in animals 30.7 ± 0.9 years old (n = 7) (p < 0.01). Similar age-related reductions in the activities of complexes I and IV were obtained when enzyme activities were corrected for complex II-III activity. These findings show an age-associated progressive impairment of mitochondrial complex I and complex IV activities in cerebral cortices of primates.     

Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

Inhibition of Bordetella pertussis filamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

Abstract Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis , is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.     

Synthesis and evaluation of the antidepressant activity of the enantiomers of bupropion

The synthesis of the enantiomers of bupropion, (rac)-2-tert-butylamino-3′-chloropropiophenone 1 (Wellbutrin®) is described. The enantiomers were compared with the racemate in both the tetrabenazine-induced sedation model and the inhibition of uptake of biogenic amine assay. No significant differences were found in their potencies to reverse tetrabenazine-induced sedation in mice or in their IC₅₀ values as inhibitors of biogenic amine uptake into nerve endings obtained from mouse brain. © 1993 Wiley-Liss, Inc.