Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

Sites of Tubulin Polymerization in PC 12 Cells

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Reductive Desulfurization of Dibenzyldisulfide

Dibenzyldisulfide was reductively degraded by a methanogenic mixed culture derived from a sewage digestor. Toluene was produced with benzyl mercaptan as an intermediate in sulfur-limited medium. Toluene production was strictly associated with biological activity; however, the reducing agent for the culture medium, Ti(III), was partially responsible for production of benzyl mercaptan. Sulfide was not detected. Additions of sodium sulfide did not inhibit toluene production. Additions of 2-bromoethane sulfonic acid prevented methanogenesis but did not adversely affect toluene yields.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Citrus limonoid effects on Colorado potato beetle larval survival and development

The effects of citrus limonoids, applied topically to potato (Solanum tuberosum L. var. Katahdin) foliage, on Colorado potato beetle (Leptinotarsa decemlineata) (Say) (Chrysomelidae) larval development, growth, and survival were quantified in laboratory assays and a small-plot field test. In laboratory assays, survival, development rate, and body weight decreased with increasing limonoid concentration, however these measures of larval response did not significantly differ among varying periods of limonoid exposure (three, six, or nine days). Significant limonoid application concentration and frequency effects on survival, development rate, and defoliation were observed in the field test. These results indicate the potential utility of lethal and non-lethal effects of citrus limonoids for management of the Colorado potato beetle.     

A microdialysis study of the effects of the nicotinic agonist RJR-2403 on cortical release of acetylcholine and biogenic amines

Transcortical dialysis was employed to investigate the effects of subcutaneous (s.c.) injections of RJR-2403 (1.2–7.2 μmol/kg) on extracellular levels of acetylcholine (ACh), norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat. Systemic administration of RJR-2403 produced a 90% increase of cortical extracellular ACh levels that persisted for up to 90 minutes after injection. Norepinephrine and DA release were increased 124% and 131% above basal values, respectively. Serotonin (5-HT) levels in the dialysate were also significantly elevated by RJR-2403 (3.6 μmol/kg, s.c.) 70% above baseline at 90 minutes post-injection. Comparison of these responses to those of (−)nicotine from a previous study reveals little difference between the two compounds in their ability to influence cortical neurotransmitter release following systemic administration.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Abstractions can be causes — A response to professor hogan

In Can Abstractions be Causes, David Johnson defends the view that abstractions can have causal force. He offers as his own example of natural kinds ecological niches, arguing that the causal force of these niches in nature is akin to the force of Aristotelian final causes. He concludes that, rooted as it is in seventeenth century mechanism, the currently-accepted model of causality which recognises only efficient causes is inadequate to the needs of contemporary science. In Natural Kinds and Ecological Niches — Response to Johnson's Paper, Melinda Hogan offers a critique of Johnson's paper which, by begging the question in favor of the very sort of causality Johnson seeks to supplement, misses the epistemological implications of his idea. In this paper I will attempt to clarify and defend Johnson's position, pointing out some of its implications for the epistemology of science in general.