Biosynthesis of high density lipoprotein by chicken liver: nature of nascent intracellular high density lipoprotein

Young chickens were administered L-[(3)H]leucine and after 10 or 30 min the livers were removed and fractioned into rough (RER) and smooth (SER) endoplasmic reticulum fractions and into light, intermediate, and heavy golgo cell fractions. The labeled high density lipoprotein (HDL), contained within these intracellular organelles was isolated either by immunoprecipitation using rabbit antiserum to rooster HDL, or by ultracentrifugal glotation between densities 1.063 and 1.21 g/ml. The radioactive apoproteins of nascent HDL were analyzed by SDS PAGE and detected by fluorography. Analyses of radioactive apoproteins obtained by immunoprecipitation from the contents of the RER, the SER, and the three golgi complex fractions revealed only one apoprotein, A1. The C peptide present in serum HDL was not detected intracellularly. The radioactive apoprotein A1 which is present within the cisternae of the RER and the SER fractions failed to float, whereas apoprotein A1, present within the golgi apparatus, readily floated between densities 1.063 and 1.21 g/ml. The HDL particles, isolated by flotation from the golgi apparatus content, were further characterized by lipid and protein analyses and by electron microscopy. Golgi HDL particles have the same density as serum HDL. On a percentage basis, golgi HDL contains less protein and more phospholipids than does serum HDL. Morphologically, golgi HDL is different in appearance from serum HDL. It is more heterogeneous in size, with most of the particles ranging 8.3-25 nm in diameter. The spherical particles contain small membrane tails. Occasionally, a few disk-shaped bilayer structures are also found within the golgi apparatus. These studies show that the newly synthesized apoprotein A1, present within the RER and the SER cell fractions, is not fully complexed with lipid and that apoprotein A1 does not acquire sufficient lipid to float at the proper HDL density until it enters the golgi apparatus. The difference in chemical composition and the heterogeneous size of golgi HDL may be attributed to the different stages of HDL maturation.     

Postsynaptic density antigens: preparation and characterization of an antiserum against postsynaptic densities

Long-term immunization of rabbits with postsynaptic densities (PSD) from bovine brain produced an antiserum specific for PSD as judged by binding to subcellular fractions and immunohistochemical location at the light and electron microscope levels. (a) The major antigens of bovine PSD preparations were three polypeptides of molecular weight 95,000 (PSD-95), 82,000 (PSD-82), and 72,000 (PSD-72), respectively. Antigen PSD-95, also present in mouse and rat PSDs was virtually absent from cytoplasm, myelin, mitochondria, and microsomes from rodent or bovine brain. Antigens PSD-82 and PSD-72 were present in all subcellular fractions from bovine brain, especially in mitochondria, but were almost absent from rodent brain. The antiserum also contained low-affinity antibodies against tubulin. (b)Immunohistochemical studies were performed in mouse and rat brain, where antigen PSD-95 accounted for 90 percent of the antiserum binding after adsorption with purified brain tubulin. At the light microscope level, antibody binding was observed only in those regions of the brain where synapses are known to be present. No reaction was observed in myelinated tracts, in the neuronal cytoplasm, or in nonneuronal cells. Strong reactivity was observed in the molecular layer of the dentate gyrus, stratum oriens and stratum radiatum of the hippocampus, and the molecular layer of the cerebellum. Experimental lesions, such as ablation of the rat entorhinal cortex or intraventricular injection of kainic acid, which led to a major loss of PSD in well- defined areas of the hippocampal formation, caused a correlative decrease in immunoreactivity in these areas. Abnormal patterns of immunohistochemical staining correlated with abnormal synaptic patterns in the cerebella of reeler and staggerer mouse mutants. (c) At the electron microscopic level, immunoreactivity was detectable only in PSD. The antibody did not bind to myelin, mitochondria or plasma membranes. (d) The results indicate that antigen PSD-95 is located predominantly or exclusively in PSD and can be used as a marker during subcellular fractionation. Other potential uses include the study of synaptogenesis, and the detection of changes in synapse number after experimental perturbations of the nervous system.     

Role of the metF and metJ genes on the vitamin B12 regulation of methionine gene expression: involvement of N5-methyltetrahydrofolic acid.

The repression of MetE synthesis in Escherichia coli by vitamin B12 is known to require the MetH holoenzyme (B12-dependent methyltransferase) and the metF gene product. Experiments using trimethoprim, an inhibitor of dihydrofolate reductase, show that the MetF protein is not directly involved in the repression, but that N5-methyltetrahydrofolic acid (N5-methyl-H4-folate), the product of the MetF enzymatic reaction is required. Since the methyl group from N5-methyl-H4-folate is normally transferred to the MetH holoenzyme to form a methyl-B12 enzyme, the present results suggest that a methyl-B12 enzyme is involved in the vitamin B12 repression of metE expression. Other results argue against the possibility that a methyl-B12 enzyme functions in this repression solely by decreasing the cellular level of homocysteine, which is required for MetR activation of metE expression. Experiments with metJ mutants show that the MetJ protein mediates about 50% of the repression of metE expression by B12 but is totally responsible for the regulation of metF expression by vitamin B12.     

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus).

1. The in vitro metabolism of [3H]benzo[a]pyrene (BP) and [14C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo. 2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of BP-4,5-diol. 3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides. 4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates. 5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25-30 days. The major adduct was anti-BPDE-deoxyguanosine.     

Ring-andN-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system.

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.     

Inactivation of the sodium channel. I. Sodium current experiments

Inactivation of sodium conductance has been studied in squid axons with voltage clamp techniques and with the enzyme pronase which selectively destroys inactivation. Comparison of the sodium current before and after pronase treatment shows a lag of several hundred microseconds in the onset of inactivation after depolarization. This lag can of several hundred microseconds in the onset of inactivation after polarization. This lag can also be demonstrated with double-pulse experiments. When the membrane potential is hyperpolarized to -140 mV before depolarization, both activation and inactivation are delayed. These findings suggest that inactivation occurs only after activation are delayed. These findings suggest that inactivation occurs only after activation; i.e. that the channels must open before they can inactivate. The time constant of inactivation measured with two pulses (τ(c)) is the same as the one measured from the decay of the sodium current during a single pulse (τ(h)). For large depolarizations, steady-state inactivation becomes more incomplete as voltage increases; but it is relatively complete and appears independent of voltage when determined with a two- pulse method. This result confirms the existence of a second open state for Na channels, as proposed by Chandler and Meves (1970. J. Physiol. [Lond.]. 211:653-678). The time constant of recovery from inactivation is voltage dependent and decreases as the membrane potential is made more negative. A model for Na channels is presented which has voltage-dependent transitions between the closed and open states, and a voltage-independent transition between the open and the inactivated state. In this model the voltage dependence of inactivation is a consequence of coupling to the activation process.     

The Effects of Anthropogenic Structures on Habitat Connectivity and the Potential Spread of Non-Native Invertebrate Species in the Offshore Environment

Offshore structures provide habitat that could facilitate species range expansions and the introduction of non-native species into new geographic areas. Surveys of assemblages of seven offshore oil and gas platforms in the Santa Barbara Channel revealed a change in distribution of the non-native sessile invertebrate Watersipora subtorquata, a bryozoan with a planktonic larval duration (PLD) of 24 hours or less, from one platform in 2001 to four platforms in 2013. We use a three-dimensional biophysical model to assess whether larval dispersal via currents from harbors to platforms and among platforms is a plausible mechanism to explain the change in distribution of Watersipora and to predict potential spread to other platforms in the future. Hull fouling is another possible mechanism to explain the change in distribution of Watersipora. We find that larval dispersal via currents could account for the increase in distribution of Watersipora from one to four platforms and that Watersipora is unlikely to spread from these four platforms to additional platforms through larval dispersal. Our results also suggest that larvae with PLDs of 24 hours or less released from offshore platforms can attain much greater dispersal distances than larvae with PLDs of 24 hours or less released from nearshore habitat. We hypothesize that the enhanced dispersal distance of larvae released from offshore platforms is driven by a combination of the offshore hydrodynamic environment, larval behavior, and larval release above the seafloor.     

MERCURY INHIBITION ON PRIMARY PRODUCTIVITY USING LARGE VOLUME PLASTIC CHAMBERS IN SITU1

Approximately 6000 l of lake water was suspended in large, clear plastic chambers in Lake Powell, Arizona. The chambers were monitored during 24 h incubation periods in the spring and summer 1974 and 1975. Physico-chemical differences between the natural lake system and within the experimental chambers were negligible. Experimental mercury concentrations ranging from < 0.02 to 1.25 ppm were added to the chambers to determine the effect of elevated Hg concentrations on in situ primary production. Mercury concentrations were monitored in the water column of each chamber during each 24 h incubation period to determine absolute concentrations following reduction through adsorption, absorption and vaporization. At least a 40% reduction in photosynthetic activity occurred at Hg concentrations as low as 0.06 ppm. A toxic Hg threshold concentration of 0.06 ppm was demonstrated for the summer phytoplankton assemblage, but a distinct threshold concentration was absent for the spring diatom assemblage. Differences in spring and summer phytoplankton populations may suggest subtle differences in Hg sensitivity between phytoplankton assemblages in combination with temperature acting on total community metabolism.