Proteolytic modification of the amino-terminal and carboxyl-terminal regions of rat hepatic phenylalanine hydroxylase

Activation of rat liver phenylalanine hydroxylase by limited proteolysis catalyzed by chymotrypsin was investigated with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure gel filtration. Both activation and proteolysis were decreased by the addition of the natural cofactor, (6R)-tetrahydrobiopterin. From chymotryptic digests of the hydroxylase carried out in the presence and absence of (6R)-tetrahydrobiopterin, several different enzyme species were isolated by high pressure gel filtration. One species (subunit Mr = 47,000) with unchanged hydroxylase activity was isolated from the chymotryptic digest in the presence of (6R)-tetrahydrobiopterin; it was derived from the native enzyme (Mr = 52,000) by cleavage of the COOH-terminal Mr = 5,000 portion of the native enzyme. In the absence of (6R)-tetrahydrobiopterin, another species (subunit Mr = 36,000) was isolated. In addition to modification at the COOH-terminal end of the molecule, this species also had lost a Mr = 11,000 fragment from the NH2-terminal end of the hydroxylase. The Mr = 11,000 fragment was shown to include the phosphorylation site of the enzyme. This Mr = 36,000 species was 30-fold more active than the native phenylalanine hydroxylase when assayed in the presence of tetrahydrobiopterin. These results suggest that the regulatory domain that inhibits hydroxylase activity in the basal state may be located at the NH2 terminus of the phenylalanine hydroxylase subunit.     

Protein kinase C: a new linkage marker for growth hormone and for COL1A1

An expanded linkage group on the long arm of human chromosome 17 is reported. Using the CEPH panel of DNAs and restriction fragment length polymorphism (RFLP) markers for the centromere locus (D17Z1), growth hormone (GH1), collagen type I alpha 1 (COL1A1), and protein kinase C-alpha polypeptide (PKCA) loci, theta values of 0.03, 0.11, and 0.23 were found between PKCA and GH1, PKCA and COL1A1, and PKCA and D17Z1, respectively. The theta values calculated for GH1 versus COL1A1 or D17Z1 were 0.11 and 0.23, respectively. Sex-specific recombination rates were calculated for the best likelihood order and demonstrate female recombination greater than male recombination. Therefore, the loci studied span a map region of approximately 30 cm between 17cen and 17q24, with the most likely gene order being D17Z1-COL1A1-PKCA-GH1.     

Determination of the enantiomeric composition of (R*,S*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate, the male-produced aggregation pheromone of Sitophilus granarius

The enantiomeric composition of sitophilate, the granary weevil [Sitophilus granarius (L.)] male-produced aggregation pheromone [(R^*,S^*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate)], was determined by three methods: (1) bioassaying the synthetic (2S,3R) and (2R,3S) enantiomers of the active (R^*,S^*) diastereomer; (2) ¹H NMR spectroscopy of Mosher ester derivatives of the natural pheromone and synthetic (2S,3R)-and (2R,3S)-sitophilate; and (3) capillary GLC comparisons of the retention times of derivatized natural pheromone and the two synthetic enantiomers. The combined methods confirmed the (2S,3R) enantiomer as the active form of sitophilate. Male granary weevils were shown to produce >96% (2S,3R)-sitophilate. No significant attraction of S. granarius by the (2R,3S) enantiomer was observed. Rice and maize weevils [S. oryzae (L.) and S. zeamais Motschulsky] were not attracted by (2S,3R)-sitophilate. S. granarius L. est un déprédateur important des grains stockés. Le (R^*,S^*)-1-éthylpropyl 2-méthyl-3-hydroxypentanoate a été identifié en 1987 comme le principal composé du sitophilate, la phéromone mâle d'agrégation de S. granarius. La composition énantiométrique du sitophilate a été déterminée par 3 méthodes:

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.     

Maturation of the rat cumulus-oocyte complex: structure and function

The cumulus cells that surround the mammalian oocyte become dispersed following the preovulatory surge of the pituitary gonadotropin, luteinizing hormone (LH). We have examined cumulus-oocyte complexes of PMSG-primed immature rats before and at 1, 2, 3, 4, 6, and 8 hr after injection of human chorionic gonadotropin (hCG), which acts on the rat ovary like the pituitary gonadotropin. Associations between projections of the cumulus cells and the oocyte were analyzed in thin sections. We observed that some cumulus projections were greatly enlarged where they associate with the oocyte. These enlarged regions were filled with numerous small vesicles. Gap junctions between cumulus cell projections and the oocytes were small. We quantitated the number and size of gap junctions between cumulus cells. The number of small gap junctions (less than 1 microM) between cumulus cells did not change significantly over the 8-hr period after hCG administration. Larger gap junctions, however, showed a general downward trend beginning after the third hour post hCG. Light microscopic observations of plastic sections revealed that dispersion of the cumulus oophorus is not observed until after 4 hr post-hCG, but between 4 and 8 hr after gonadotropin administration the cumulus becomes markedly dispersed. In the majority of the oocytes in these complexes the germinal vesicle (GV) displayed some irregularity in shape at 2 hr post-hCG, although absence of the GV was not observed until later. Our observations suggest a new means of communication in the cumulus-oocyte complex by the vesicle-filled enlargements of the cumulus cell projections at the oocyte surface. They further indicate that the decrease in metabolic coupling observed in rat cumulus-oocyte complexes soon after exposure to LH is not associated with a change in number and size of the gap junctions between the cumulus cells. We suggest that it is either the disruption of the gap junctions at the region of contact of the cumulus cell projections with the oocyte surface or the operation of a gating mechanism that blocks the junctional channels without affecting their morphological appearance that is responsible for uncoupling of the oocyte from the cumulus cells.     

Measurement of bacterial random motility and chemotaxis coefficients: I. Stopped-flow diffusion chamber assay

Bacterial chemotaxis, the directed movement of a cell population in response to a chemical gradient, plays a critical role in the distribution and dynamic interaction of bacterial populations in nonmixed systems. Therefore, in order to make reliable predictions about the migratory behavior of bacteria within the environment, a quantitative characterization of the chemotactic response in terms of intrinsic cell properties is needed.The design of the stopped-flow diffusion chamber (SFDC) provides a well-characterized chemical gradient and reliable method for measuring bacterial migration behavior. During flow through the chamber, a step change in chemical concentration is imposed on a uniform suspension of bacteria. Once flow is stopped, diffusion causes a transient chemical gradient to develop, and bacteria respond by forming a band of high cell density which travels toward higher concentrations of the attractant. Changes in bacterial spatial distributions observed through light scattering are recorded on photomicrographs during a 10-min period. Computer-aided image analysis converts absorbance of the photographic negatives to a digital representation of bacterial density profiles. A mathematical model (part II) is used to quantitatively characterize these observations in terms of intrinsic cell parameters: a chemotactic sensitivity coefficient, mu(0), from the aggregate cell density accumulated in the band and a random motility coefficient, mu, from population dispersion in the absence of a chemical gradient.Using the SFDC assay and an individual-cell-based mathematical model, we successfully determined values for both of these population parameters for Escherichia coli K12 responding to fucose. The values obtained were mu = 1.1 +/- 0. 4 x 10(-5) cm(2)/s and chi(o) = 8 +/- 3 +/- 10(-5) cm(2)/s. We have demonstrated a method capable of determining these parameter values from the now validated mathematical model which will be useful for predicting bacterial migration in application systems.     

Screening of a broad range of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) germplasm for <Emphasis Type="Italic">in vitro</Emphasis> rapid plant regeneration and development of an early prediction system

Rice has emerged as a model monocot for studies in agriculture and biotechnology due to its relatively small genome and a ready accessibility to plant material. Tissue culture is one of the tools required for genetic transformation and some breeding programs, and the selection of high-frequency regenerator types is essential for success in these technologies. Thirty-three rice entries with agricultural and biotechnological characteristics of interest were screened with the aim to identify the best regenerators. Entries that exhibited between 50% and 90% regeneration frequencies include ‘Taipei-309,’ ‘Super Dwarf,’ ‘Norin’ (japonica types), PI 312777, ‘Ali Combo’ (indica types), ‘STG-S,’ and ‘LA3’ (red rice types). One third of the entries tested were at least two times better at regeneration than the often-cited regenerator ‘Nipponbare.’ Those entries showing at least 85% frequency of greening or somatic embryo formation at 15 or 30 d on regeneration medium ultimately produced whole plants after 45 d on regeneration medium at high frequency (at least 40%); those entries not reaching the 85% threshold of greening by Days 15 or 30 exhibited moderate (15–40%) to low (less than 10%) frequency of whole plant regeneration. This greening response suggests the means for an early prediction system for identification of useful rice regenerator lines, which would be beneficial for high-throughput screening of germplasm as well as for decreasing the time and cost of in vitro culture.