The molecular basis of mutations at the Waxy locus from Amaranthus caudatus L.: evolution of the waxy phenotype in three species of grain amaranth

Polymorphisms at the Waxy locus of Amaranthus caudatus L. collected from a wide range of regions were used to investigate genetic diversity and mutation sites. A comparison of the Waxy locus revealed a very high level of sequence conservation. This result clearly showed low environmental and evolutionary variability in the Waxy gene. We also performed screening to confirm the mutation sites in the coding sequences of all accessions. The results indicate that one insertion in the coding region of Waxy genes was responsible for the change in perisperm starch leading to the waxy phenotype in all accessions of this species, and thus that a single mutation event altered the regulation of the Waxy gene during the domestication of this crop. In addition, phylogenetic analysis showed that waxy phenotypes within each of three species, A. caudatus, A. cruentus and A. hypochondriacus, originated separately or differentiated from nonwaxy phenotypes of each species through a single mutational event (i.e., a frame shift or base substitution). We also compared obvious structural features of the coding sequence of waxy and nonwaxy phenotypes with those of low-amylose phenotypes in A. caudatus. The Waxy coding sequences of low-amylose phenotypes do not show polymorphisms and are identical with those of waxy phenotypes. This could mean that there is another gene that encodes a key enzyme responsible for amylose synthesis as the elementary quantity in tissues other than perisperm in A. caudatus.     

Records ofApristurus herklotsi (Lamniformes,Scyliorhinidae) and discussion of its taxonomic relationships

Additional specimens of the rareApristurus herklotsi are reported, and the characteristic features of this species are discussed.A. herklotsi is concluded to be a distinct species, having a very long snout, a narrow distance between the nostrils, a long caudal fin, a short abdomen, numerous teeth on both jaws, and a low number of monospondylous vertebrae.A. herklotsi appears to be mature at about 44 cm in total length.     

Wiring diagrams of MAPK regulation by MEKK1, 2, and 3.

Mitogen-activated protein kinase (MAPK) pathways are activated by a plethora of stimuli. The literature is filled with papers describing the activation of different MAPKs by almost any stimulus or insult imaginable to cells. In this review, we use signal transduction wiring diagrams to illustrate putative upstream regulators for the MAPK kinase kinases, MEKK1, 2, and 3. Targeted gene disruption of MEKK1, 2, or 3 defined phenotypes for each MEKK associated with loss of specific MAPK regulation. Genetic analysis of MEKK function clearly defines specific components of the wiring diagram that require MEKK1, 2, or 3 for physiological responses. We propose that signal transduction network wiring diagrams are valuable tools for hypothesis building and filtering physiologically relevant phenotypic responses from less connected protein relations in the regulation of MAPK pathways.     

Oxygen transfer properties and dimensions of red blood cells in high-altitude camelids,dromedary camel and goat

Summary To estimate the advantage of the small red blood cells (RBC) of high-altitude camelids for O₂ transfer, the kinetics of O₂ uptake into and release from the RBC obtained from llama, vicuña and alpaca were investigated at 37°C with a stopped-flow technique. O₂ transfer conductance of RBC (G) was estimated from the rate of O₂ saturation change and the corresponding O₂ pressure difference between medium and hemoglobin. For comparison, O₂ kinetics for the RBC of a lowaltitude camelid (dromedary camel) and the pygmy goat were determined and previously measured values for human RBC were used. O₂ transfer of RBC was found to be strongly influenced by extracellular diffusion, except with O₂ release into dithionite solutions of sufficiently high concentration (>30 mM). TheG values measured in these standard conditions,G _st (in mmol · min^–1 · Torr^–1 · (ml RBC)^–1) were: high-altitude camelids, 0.58 (averaged for llama, alpaca and vicuña since there were no significant interspecific differences); camel 0.42; goat, 0.42; man, 0.39. The differences can in part be attributed to expected effects of the size and shape of the RBC (volume, surface area, mean thickness), as well as to the intracellular O₂ diffusivity which depends on the concentration of cellular hemoglobin. The highG _st of RBC of highaltitude camelids may be considered to enhance O₂ transfer in lungs and tissues. But the O₂ transfer conductance of blood, , equal toG _st multiplied by hematocrit (in mmol · min^–1 · Torr^–1 · (ml blood)^–1), was only slightly higher as compared to other species: 0.20 (llama, alpaca, vicuña), 0.14 (camel), 0.18 (goat), 0.17 (man).Abbreviations DPG 2,3-diphosphoglycerate - G conductance - Hb hemoglobin - RBC red blood cells - percent saturation of hemoglobin     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Natural development time of Eodiaptomus japonicus (Copepoda: Calanoida) in Lake Biwa

Seasonal and ontogenetic changes in natural development timewere studied for Eodiaptomus japonicus in Lake Biwa in 1986and 1987. Wild individuals in a certain developmental stagewere collected, fed on natural food and examined until theyhad moulted twice. Natural development times fluctuated irrespectiveof temperature from May to October. Food deficiency delayeddevelopment in all feeding stages, and food availability probablydetermined natural development time. Serious food limitationraised mortality in copepodid stage I. In November developmentwas delayed even with enough food. The development of E.japonicuswas almost isochronal except for a short prefeeding naupliarstage I and a long copepodid stage V.     

Protective effects of glutathione against lipid peroxidation in chronically iron-loaded mice

To elucidate the protective effects of glutathione against iron-induced peroxidative injury, changes in the hepatic glutathione metabolism were studied in chronically iron-loaded mice. When the diets of the mice were supplemented with carbonyl iron, iron deposition occurred primarily in the parenchymal cells of the liver. In addition, expiratory ethane production was elevated, suggesting an enhancement in lipid peroxidation. In iron-loaded mice, the total hepatic glutathione contents were higher (6.21 +/- 0.53 mumol/g wet wt.) than in control mice (4.61 +/- 0.31 mumol/g wet wt.), primarily due to an increase in the reduced glutathione contents. The value of oxidized glutathione was also higher (98.5 +/- 8.1 nmol/g wet wt.) than in the controls (60.8 +/- 9.5 nmol/g wet wt.), and the ratio of oxidized glutathione to total glutathione increased. The excretion rate of glutathione from the hepatocytes in iron-loaded mice also increased. These observations suggest that chronic iron-loading of mice stimulates lipid peroxidation and oxidation of glutathione and that peroxidized molecules may be catabolized using reduced glutathione.     

Several new substrates for Desulfovibrio vulgaris strain Marburg and a spontaneous mutant from it

The ability of Desulfovibrio vulgaris strain Marburg (DSM 2119) to oxidize alcohols was surveyed in the presence and absence of hydrogen-scavenging anaerobes, Acetobacterium woodii and Methanospirillum hungatei. In the presence of sulfate, D. vulgaris grew not only on ethanol, 1-propanol, and 1-butanol, but also on isobutanol, 1-pentanol, ethyleneglycol, and 1,3-propanediol. Metabolism of these alcohols was simple oxidation to the corresponding acids, except with the last two substrates: ethyleneglycol was oxidized to glycolate plus acetate, 1,3-propanediol to 3-hydroxypropionate plus acetate. Experimental evidence was obtained, suggesting that 2-methoxyethanol was not utilized by all the cells of strain marburg, but by a spontaneous mutant. 2-Methoxyethanol was oxidized to methoxyacetate by the mutant. Co-culture of strain Marburg plus A. woodii grew on ethanol, 1-propanol, 1-butanol, and 1,3-propanediol in the absence of sulfate. Co-culture of strain Marburg plus M. hungatei grew on ethanol, 1-propanol, and 1-butanol, but not on ethyleneglycol and 1,3-propanediol, Co-culture of the mutant plus A. woodii or M. hungatei did not grow on 2-methoxyethanol.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-α, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-α mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-α protein by DLD-1 and CoLo320DM cells was confirmed by TGF-α specific monoclonal antibody binding assay. The spontaneous³H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-α monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-α produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.