The effects of Rubisco activase on C4 photosynthesis and metabolism at high temperature.

The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme facilitates the release of sugar phosphate inhibitors from Rubisco catalytic sites thereby influencing carbamylation. T(1) progeny of transgenic Flaveria bidentis (a C(4) dicot) containing genetically reduced levels of Rubisco activase were used to explore the role of the enzyme in C(4) photosynthesis at high temperature. A range of T(1) progeny was screened at 25 degrees C and 40 degrees C for Rubisco activase content, photosynthetic rate, Rubisco carbamylation, and photosynthetic metabolite pools. The small isoform of F. bidentis activase was expressed and purified from E. coli and used to quantify leaf activase content. In wild-type F. bidentis, the activase monomer content was 10.6+/-0.8 micromol m(-2) (447+/-36 mg m(-2)) compared to a Rubisco site content of 14.2+/-0.8 micromol m(-2). CO(2) assimilation rates and Rubisco carbamylation declined at both 25 degrees C and 40 degrees C when the Rubisco activase content dropped below 3 mumol m(-2) (125 mg m(-2)), with the status of Rubisco carbamylation at an activase content greater than this threshold value being 44+/-5% at 40 degrees C compared to 81+/-2% at 25 degrees C. When the CO(2) assimilation rate was reduced, ribulose-1,5-bisphosphate and aspartate pools increased whereas 3-phosphoglycerate and phosphoenol pyruvate levels decreased, demonstrating an interconnectivity of the C(3) and C(4) metabolites pools. It is concluded that during short-term treatment at 40 degrees C, Rubisco activase content is not the only factor modulating Rubisco carbamylation during C(4) photosynthesis.

Reductions of Rubisco activase by antisense RNA in the C4 plant Flaveria bidentis reduces Rubisco carbamylation and leaf photosynthesis.

To function, the catalytic sites of Rubisco (EC 4.1.1.39) need to be activated by the reversible carbamylation of a lysine residue within the sites followed by rapid binding of magnesium. The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme is thought to aid the release of sugar phosphate inhibitors from Rubisco's catalytic sites, thereby influencing carbamylation. In C3 species, Rubisco operates in a low CO2 environment, which is suboptimal for both catalysis and carbamylation. In C4 plants, Rubisco is located in the bundle sheath cells and operates in a high CO2 atmosphere close to saturation. To explore the role of Rubisco activase in C4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C4 dicot, by transformation with an antisense gene directed against the mRNA for Rubisco activase. Four primary transformants with very low activase levels were recovered. These plants and several of their segregating T1 progeny required high CO2 (>1 kPa) for growth. They had very low CO2 assimilation rates at high light and ambient CO2, and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO2. The amount of Rubisco was similar to that of wild-type plants. Experiments with the T1 progeny of these four primary transformants showed that CO2 assimilation rate and Rubisco carbamylation were severely reduced in plants with less than 30% of wild-type levels of activase. We conclude that activase activity is essential for the operation of the C4 photosynthetic pathway.

Achievement of ultralow emittance beam in the accelerator test facility damping ring.

For high luminosity in electron-positron linear colliders, it is essential to generate low vertical emittance beams. We report on the smallest vertical emittance achieved in single-bunch-mode operation of the Accelerator Test Facility, which satisfies the requirement of the x-band linear collider. The emittances were measured with a laser-wire beam-profile monitor installed in the damping ring. The bunch length and the momentum spread of the beam were also recorded under the same conditions. The smallest vertical rms emittance measured at low intensity is 4 pm at a beam energy of 1.3 GeV, which corresponds to the normalized emittance of 1.0x1.0(-8) m. It increases by a factor of 1.5 for a bunch intensity of 10(10) electrons. The measured data agreed to the calculation of intrabeam scattering within much better than a factor of 2.

Lovastatin biosynthesis in Aspergillus terreus: characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene.

BACKGROUND: Lovastatin, an HMG-CoA reductase inhibitor produced by the fungus Aspergillus terreus, is composed of two polyketide chains. One is a nonaketide that undergoes cyclization to a hexahydronaphthalene ring system and the other is a simple diketide, 2-methylbutyrate. Fungal polyketide synthase (PKS) systems are of great interest and their genetic manipulation should lead to novel compounds. RESULTS: An A. terreus mutant (BX102) was isolated that could not synthesize the nonaketide portion of lovastatin and was missing a approximately 250 kDa polypeptide normally present under conditions of lovastatin production. Other mutants produced lovastatin intermediates without the methylbutyryl sidechain and were missing a polypeptide of approximately 220 kDa. The PKS inhibitor cerulenin reacted covalently with both polypeptides. Antiserum raised against the approximately 250 kDa polypeptide was used to isolate the corresponding gene, which complemented the BX102 mutation. The gene encodes a polypeptide of 269 kDa containing catalytic domains typical of vertebrate fatty acid and fungal PKSs, plus two additional domains not previously seen in PKSs: a centrally located methyltransferase domain and a peptide synthetase elongation domain at the carboxyl terminus. CONCLUSIONS: The results show that the nonaketide and diketide portions of lovastatin are synthesized by separate large multifunctional PKSs. Elucidation of the primary structure of the PKS that forms the lovastatin nonaketide, as well as characterization of blocked mutants, provides new details of lovastatin biosynthesis.

Morbidity on Kauai before and after Hurricane Iniki.

BACKGROUND: On September 11, 1992, Hurricane Iniki, a Class III/IV storm, passed directly over Kauai. This study is the first attempt to measure increases in injuries and other health outcomes among an entire population in the impact zone of a hurricane. METHODS: Medical chart data were abstracted from all facilities providing primary and emergency care on Kauai. Incidence of injury, cardiovascular disease, and asthma for the 2-week period following Hurricane Iniki were compared to those for the 2-week period preceding Iniki. RESULTS: A total of 1,584 injuries were treated in the post-Iniki period compared with 231 injuries treated in the pre-Iniki period (relative risk = 6.86, 95% confidence interval 5.98-7.87). Open wounds constituted over half of these injuries. Physician visits for asthma and cardiovascular disease were also significantly increased in the post-Iniki period (relative risks, respectively: 2.81, 95% confidence interval 1.93-4.09; 2.73, 95% confidence interval 1.51-4.94). CONCLUSIONS: Significant increases in the incidence of injuries, asthma, and cardiovascular disease occurred following Hurricane Iniki. Although no changes occurred in the proportion of patients needing hospitalization, additional injuries and illnesses after a natural disaster can burden existing medical facilities in a rural community with limited resources. Disaster preparedness plans need to include methods to increase services and supplies at existing medical facilities.

Mortality of Kauai residents in the 12-month period following Hurricane Iniki.

On September 11, 1992, Hurricane Iniki struck Kauai leaving all residents without electricity and telephone services and damaging 70% of the homes. This study examined the hypothesis that Hurricane Iniki increased the mortality of Kauai residents by comparing mortality data for the 5 years preceding Hurricane Iniki with mortality data for the 12 months immediately following. Although the overall mortality rate was increased in the post-Iniki period, the only significant increase was in the rate of diabetes mellitus-related deaths (relative risk = 2.61, 95% confidence interval 1.44-4.74). Hurricane Iniki did not appear to significantly increase the risk of dying of Kauai residents in the 12 months immediately following the disaster.

The appearance of rod opsin during monkey retinal development.

PURPOSE: To determine the temporal and spatial pattern of rod opsin appearance in Macaca monkey retina. METHODS: Frozen sections from fetal day (Fd) 55 to adulthood (birth = Fd168) containing the entire horizontal meridian were stained using Rho4D2 monoclonal antiserum visualized with immunofluorescent labeling. At Fd66, Fd79, and Fd89, retinal samples taken at known eccentricities were studied from the opposite eye using standard electron microscope methods. RESULTS: Rod opsin was detected at Fd66 in or near the fovea, and a second focus appeared at Fd75 to Fd77 near the optic disc in the nasal rod ring. The earliest opsin appeared in the apical stubs, which resembled the apical connecting cilium in the electron microscope. Staining of the entire cell body membrane, including the synaptic spherule, was present 4 to 7 days later. Opsin expression had a nasal bias with rods at the nasal ora labeled at Fd140, whereas temporal ora was not labeled until Fd155. Cell body labeling disappeared by Fd132 across central retina but persisted into the first postnatal year in far peripheral retina. Outer segment (OS) length measurements showed that rods in the rod ring had the longest OS between Fd115 and postnatal week 9. Rod OS at all retinal eccentricites continued to elongate between 11 months of age and adulthood. CONCLUSIONS: Rod opsin expression follows a foveal-to-peripheral gradient beginning at Fd66 and ending near birth. Rod opsin is detected first in the connecting cilium and slightly later in the entire cell membrane, and then cell membrane labeling disappears as the heavily labeled OS elongates. Although the first OS appear on rods near the fovea, these OS still are short at birth and do not reach adult length until after 2 years of age. The longest OS at birth are found on rods at the rod ring, suggesting that this region could have higher scotopic sensitivity than central retina at birth.

Immunocytochemical localization of GABA and glycine in amacrine and displaced amacrine cells of macaque monkey retina.

Recent studies have varied widely in the percentages of GABA- and glycine-immunoreactive (GABA+, GLY+) amacrines reported for primate retina. We compared the distributions of GABA+ and GLY+ amacrines and displaced amacrines at seven locations along the horizontal meridian of macaque retina using postembedding immunogold labeling with silver intensification. The percentage of GABA+ amacrine profiles was higher in central retina (50-55%) than peripheral retina (30-40%), whereas the percentage of GLY+ amacrine profiles did not vary much with eccentricity (52-57%). GABA and glycine were colocalized in 5-20% of amacrines, depending on the eccentricity, whereas 5-30% of amacrines were not immunoreactive for either neurotransmitter. GABA+ amacrines were slightly larger than GLY+ amacrines or Müller cells. In the ganglion cell layer, 5-20% of neurons were labeled for either GABA or glycine and were identified as displaced amacrines. Of these, 53% were GABA+ only, 11% were GLY+ only, and 37% were double-labeled. A few large, very lightly labeled GABA+ cells were identified as ganglion cells. Other features that varied with eccentricity included the linear density of GABA+ and GLY+ amacrines, and the ratio of amacrines to Müller cells.

Differentiation of BC3H1 and primary skeletal muscle cells and the activity of their endogenous insulin-degrading enzyme are inhibited by the same metalloendoprotease inhibitors.

Upon reduction of serum in their media, mouse BC3H1 muscle cells withdraw from the cell cycle and begin to differentiate. In differentiating cells, the induction of muscle-specific genes is accompanied by a distinct morphological chance. However, differentiated BC3H1 cells do not fuse with each other; they remain mononucleated. Metalloendoprotease inhibitors selectively block the differentiation of BC3H1 cells while inhibitors of other protease types are ineffective. In these cells, the degradation of the internalized insulin is initiated by a 110 kDa, non-lysosomal protease known as the insulin-degrading enzyme. The same metalloendoprotease inhibitors that block BC3H1 differentiation also inhibit, with a similar specificity and potency, the in vitro and the in vivo degradation of insulin by the insulin-degrading enzyme. When the serum in the medium is reduced, the activity of the insulin-degrading enzyme in the cell cytoplasm increases rapidly. This increase precedes any detectable change in the differentiation state of these cells by about 12 hours. These results, together with very similar ones obtained with primary rat skeletal muscle cells, support our earlier proposal that the insulin-degrading enzyme is the metalloendoprotease involved in the initiation of the morphological and biochemical differentiation of muscle cells in culture.

Placental anticoagulant proteins: isolation and comparative characterization four members of the lipocortin family.

Previously we isolated and characterized a placental anticoagulant protein (PAP or PAP-I), which is a Ca2+-dependent phospholipid binding protein [Funakoshi et al. (1987) Biochemistry 26, 5572] and a member of the lipocortin family [Funakoshi et al. (1987) Biochemistry 26, 8087]. In this study, three additional anticoagulant proteins (PAP-II, PAP-III, and PAP-IV) were simultaneously isolated from human placental homogenates prepared in the presence of 5 mM ethylenediaminetetraacetic acid. The isoelectric points of PAP-I, PAP-II, PAP-III, and PAP-IV were 4.8, 6.1, 5.9, and 8.1, respectively, and their apparent molecular weights were 32,000, 33,000, 34,000, and 34,500, respectively. Amino acid sequences of cyanogen bromide fragments of these proteins showed that PAP-III was a previously unrecognized member of the lipocortin family, while PAP-II was probably the human homologue of porcine protein II and PAP-IV was a derivative of lipocortin II truncated near the amino terminus. Comparative studies showed that all four proteins inhibited blood clotting and phospholipase A2 activity with potencies consistent with their measured relative affinities for anionic phospholipid vesicles. However, PAP-IV bound to phospholipid vesicles approximately 160-fold more weakly than PAP-I, while PAP-II and PAP-III bound only 2-fold and 3-fold more weakly. These results increase to six the number of lipocortin-like proteins known to exist in human placenta. The observed differences in phospholipid binding may indicate functional differences among the members of the lipocortin family despite their considerable structural similarities.

Primary structure of human placental anticoagulant protein.

The primary structure of human placental anticoagulant protein was determined by a combination of amino acid and nucleotide sequencing techniques. The carboxymethylated protein was digested with cyanogen bromide, and the resulting peptides were separated by gel filtration and high-performance liquid chromatography. A total of 239 out of 319 amino acid residues were identified from 7 cyanogen bromide fragments. A full-length cDNA clone encoding placental anticoagulant protein was isolated from a human placenta cDNA library. This clone was 1.6 kilobases long and contained a translation initiation site coding for methionine, 957 nucleotides encoding for the mature protein, a stop codon, a poly(A) recognition site, and a poly(A) tail. Analysis of the tryptic-blocked peptide that originated from the NH2-terminus of the protein showed that the terminal methionine was removed and the adjacent alanine residue was acetylated by posttranslational events. Placental anticoagulant protein is composed of 319 amino acids with acetylalanine as the NH2-terminus and has a high degree of sequence identity with lipocortins I and II. It contains four internal repeats, each including a sequence corresponding to a putative Ca2+-dependent phospholipid binding site. Placental anticoagulant protein is a member of the lipocortin/calpactin family.

Human placental anticoagulant protein: isolation and characterization.

An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.

Amino acid sequence of the a subunit of human factor XIII.

Factor XIII is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The complete amino acid sequence of the a subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gtll cDNA library prepared from human placenta mRNA was screened with an affinity-purified antibody against the a subunit of human factor XIII and then with a synthetic oligonucleotide probe that coded for a portion of the amino acid sequence present in the activation peptide of the a subunit. Six positive clones were identified and shown to code for the a subunit of factor XIII by DNA sequence analysis. A total of 3831 base pairs was determined by sequencing six overlapping cDNA clones. This DNA sequence contains a 5' noncoding region or a region coding for a portion of a pro-piece or leader sequence, the mature protein (731 amino acids), a stop codon (TGA), a 3' noncoding region (1535 nucleotides), and a poly(A) tail (10 nucleotides). When the a subunit of human factor XIII was digested with cyanogen bromide, 11 peptides were isolated by gel filtration and reverse-phase HPLC. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 363 amino acid residues were identified. These amino acid sequences were in complete agreement with those predicted from the cDNA. The a subunit of factor XIII contained the active site sequence of Tyr-Gly-Gln-Cys-Trp, which is identical with that of tissue transglutaminase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with plasma prekallikrein.

A lambda gtll cDNA library prepared from human liver poly(A) RNA has been screened with affinity-purified antibody to human factor XI, a blood coagulation factor composed of two identical polypeptide chains linked by a disulfide bond(s). A cDNA insert coding for factor XI was isolated and shown to contain 2097 nucleotides, including 54 nucleotides coding for a leader peptide of 18 amino acids and 1821 nucleotides coding for 607 amino acids that are present in each of the 2 chains of the mature protein. The cDNA for factor XI also contained a stop codon (TGA), a potential polyadenylation or processing sequence (AACAAA), and a poly(A) tail at the 3' end. Five potential N-glycosylation sites were found in each of the two chains of factor XI. The cleavage site for the activation of factor XI by factor XIIa was identified as an internal peptide bond between Arg-369 and Ile-370 in each polypeptide chain. This was based upon the amino acid sequence predicted by the cDNA and the amino acid sequence previously reported for the amino-terminal portion of the light chain of factor XI. Each heavy chain of factor XIa (369 amino acids) was found to contain 4 tandem repeats of 90 (or 91) amino acids plus a short connecting peptide. Each repeat probably forms a separate domain containing three internal disulfide bonds. The light chains of factor XIa (each 238 amino acids) contain the catalytic portion of the enzyme with sequences that are typical of the trypsin family of serine proteases. The amino acid sequence of factor XI shows 58% identity with human plasma prekallikrein.

The structure of the carapace and plastron of juvenile turtles, Chelonia mydas (the green turtle) and Caretta caretta (the loggerhead turtle).

Consistent with their primary function as a protective covering, the carapace and plastron are heavily keratinised. In both species, the carapace is heavily pigmented and during the development and translocation of basal cells from the germinal layer of the epidermis, pigment granules migrate towards the surface layers. The epidermis is generally 2-4 cells thick; however at the growing points it can attain 6 cell layers. The epidermis is much thicker over the plastron of the loggerhead turtle. The ultrastructure of the epidermal cells supports the observation that the keratin scales are of the hard variety and the microfolds which characterise the scutes covering the carapace are discussed in relation to the lowering of frictional drag in water.

Isolation of a novel glycoprotein (EDC1) from the urine of a patient with acute myelocytic leukemia.

Gel filtration of urine from Patient ED with acute myelocytic leukemia showed a prominent protein peak with elution position corresponding to molecular weights of 20,000 to 35,000. The protein (EDC1) was isolated in pure form by sequential gel filtration and ion-exchange chromatography. Molecular weight of purified EDC1 was 27,000; it contained 27% carbohydrate and was rich in half-cystine (5% of residues). EDC1 was antigenically and chemically distinct from the recognized glycoproteins of normal plasma. With a specific rabbit antiserum and 125l-labeled EDC1, a radioimmunoassay for the glycoprotein was developed. Both noncancer and cancer plasmas contained immunoreactive material. In noncancer plasma, all the immunoreactivity was eluted from Sephadex G-75 and G-200 in position corresponding to molecular weights of 60,000 to 100,000 (Peak 1). In cancer plasma, an additional peak of immunoreactivity was eluted in the position corresponding to EDC1 (M.W., 20,000 to 30,000; Peak 2). Eighty-six % of urines from patients without clinical cancer were nonreactive in radioimmunoassay (less than 0.1 microgram immunoreactive EDC1 per ml); 11 and 3%, respectively, contained immunoreactivity equivalent to 0.1 to 0.9 and 1 to 9 microgram EDC1 per mi, entirely of Peak 1 type. Ninety-one % of urines from patients with disseminated cancer contained immunoreactivity equivalent to 10 to 9,999 microgram EDC1 per ml, primarily of Peak 2 type.