6(5)CARBOXYFLUORESCEIN AS A TRACER OF PHLOEM SAP TRANSLOCATION

6(5)carboxyfluorescein (6(5)CF), a polar fluorescein with an apparent pK of 6.3, was introduced, as a pH 6.3 solution, into the apoplast of lamina or petioles of mature soybean leaves. Freehand sections were prepared at various times and immediately observed with a fluorescence microscope. 6(5)CF-associated fluorescence appeared in all sink organs, from shoot apex to roots. It was strictly confined to the phloem regions, even after 4 days. Its transport into young leaves ceased at approximately the time they underwent sink-to-source transition. It was never transported between two leaflets of the same leaf. Its transport was interrupted by phloem destruction. All these transport characteristics were highly reproducible, and were paralleled by those of ¹⁴C transport after application of (¹⁴C)sucrose to leaf surfaces. In contrast with 6(5)CF, fluorescein was transported between mature leaves, and between leaflets of the same leaf. It was not restricted to phloem, and often appeared in the xylem region. These results indicate that 6(5)CF can be used to monitor phloem sap translocation in real time, in short- and long-term experiments.     

Locations of the hydrogenases of Methanobacterium formicicum after subcellular fractionation of cell extract.

The F420 hydrogenase of Methanobacterium formicicum was associated with membranes isolated by sucrose density gradient ultracentrifugation of cell extract. The methyl viologen hydrogenase was present in the soluble fractions. Column chromatography with phenyl-Sepharose CL-4B revealed that the F420 hydrogenase was strongly hydrophobic, suggesting that it associates with isolated membranes through hydrophobic interactions.     

Evidence of an insulin generated pyruvate dehydrogenase stimulating factor in rat brain plasma membranes

1. The results of this study indicates that the binding of insulin to brain plasma membranes activates a membrane protease which, by a trypsin like mechanism, produces a soluble factor that modulates the PDH behaviour when added to brain mitochondria. 2. The supernatant from brain plasma membranes incubated with 0.5 mg/ml trypsin added to mitochondria increases PDH activity levels and cancels PDH inhibition by NaF, as has already been seen when the plasma membranes are incubated with 25 microU/ml insulin. No such effects are obtained when the incubation is run out with 0.5 mg/ml chymotrypsin. 3. The supernatants from insulin or trypsin treated plasma membranes retain their activating properties on mitochondrial PDH also after dansylation; from these preparations a dansylated active on PDH material was separated by monodimensional chromatography on HPTLC silica Gel plates, using chloroform/1-butanol (93:7 v/v) as a solvent. 4. Insulin incubation of plasma membranes pretreated with protease inhibitors (leupeptin, phenylmethylsulfonylfluoride) or with exogenous trypsin, but not chymotrypsin substrates (esters of arginine and tyrosine) yields an inactive supernatant on PDH. 5. Insulin treated plasma membrane supernatants lose all stimulating properties on PDH after incubation for 1 hr with 2 mg/ml trypsin or chymotrypsin.     

Ultrastructural immunocytochemical study of the massa caudalis of the subcommissural organ-Reissner's fiber complex in lamprey larvae (Geotria australis): Evidence for a terminal vascular route of secretory material

Summary The massa caudalis of the subcommissural organ-Reissner's fiber complex of lamprey larvae (Geotria australis) was studied immunocytochemically at the ultrastructural level by use of the immunoperoxidase-silver methenamine procedure. An antiserum raised against bovine Reissner's fiber was utilized as primary antibody.The caudalmost portion of the central canal and its ampulla caudalis communicate, via wide intercellular spaces in their dorsal wall, with large cavities or lacunae. In addition, distinct openings in the dorsal wall of the ampulla establish an open communication between the latter and the lacunae. The lacunae are lined by slender processes of cells of unknown nature. No junctional complexes can be observed between these cells, which lack a basal lamina. The lacunae communicate with structures resembling blood capillaries, however, they are devoid of a basal lamina. These peculiar vessels, in turn, are in direct communication with characteristic blood capillaries.Reissner's fiber (RF) and its massa caudalis are strongly immunoreactive with the antiserum used. The wide intercellular spaces in the dorsal wall of the central canal and the ampulla, as well as the lumina of the (i) lacunae, (ii) modified vessels and (iii) blood capillaries are filled with a flocculent, strongly immunoreactive material. No immunoreactive material was found outside these structures. Thus, the blood capillaries appear to represent the only final target of RF-material arriving at the ampulla caudalis.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile, and Grant 6027 from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile. The authors express their gratitude to Mrs. Elizabeth Santibáñez and Mr. Julio Lamilla for providing the lamprey larvae and to Mr. Humberto Molina for preparing the three-dimensional drawing     

Small-angle X-ray scattering studies of fibrin film: comparisons of fine and coarse films prepared with thrombin and ancrod

Measurements of small-angle x-ray scattering have been made on films prepared from fine and coarse (i.e., formed at high and low, respectively, pH and ionic strength) clots of bovine fibrin by osmotic shrinkage or compression in one dimension. Intensity profiles were obtained with pinhole geometry on films stretched up to a stretch ratio of 1.43. In unstretched coarse films, repeat spacings were seen at about 245, 120, and 77-80 A. These peaks can probably be identified with the first, second, and third orders of the well-known fibrin repeat of 225 A. In unstretched fine films, only the 77-80 A spacing was seen. In this case, the first two orders may be weak because the half-staggered arrangement of monomer units giving rise to the 225 A reflection is not reinforced by lateral aggregation of protofibrils; the third order may be strong since the molecular subdomains appear to divide the repeat roughly into thirds. After stretching, the 77-80 A spacing persisted in the meridional direction but almost disappeared in the equatorial. Experiments on unstretched films prepared with ancrod substituted for thrombin gave similar results.     

On choice of substrate and habitat in brachionid rotifers

Information on the distribution of 28 rotifers of the family Brachionidae from diverse waters in south and central Sweden was analyzed to reveal their relationships to substrate and habitat. Some brachionids are preferably planktic, others periphytic and/or benthic. Some non-planktic habitats are utilized more than others, but there is no evidence of a chemical attraction from any substrate. Instead, some substrates seem to be avoided, possibly depending on a poorer flora of periphytic algae. Besides substrate type, the following factors are found to be important for creating separate ecological niches in the brachionid family: temperature, oxygen content, trophic degree, chemical environment, food choice and sensitivity to predation. It is possible to delineate separate ecological niches for all brachionid rotifers, implying that Hutchinson's ideas about the plankton paradox are contradicted. Some species are specialists, other are generalists, the latter being characterized by a great morphological variation. The species are adapted in different ways to their preferential habitats, as regards foot, egg-carrying, protrusions and other lorical structures etc. Longer spines, for instance, are generally found in more transparent water, being a supposed protection against visual predators.     

Biosynthesis of coenzyme F420 and methanopterin in Methanobacterium thermoautotrophicum

Growing cultures of Methanobacterium thermoautotrophicum were supplemented with [U-¹⁴C]adenosine or [1-¹⁴C]adenosine. 7,8-Didemethyl-8-hydroxy-5-deazariboflavin (factor F₀) and 7-methylpterin were isolated from the culture medium. Hydrolysis of cellular RNA yielded purine and pyrimidine nucleotides. The ribose side chain of proffered adenosine is efficiently incorporated into cellular adenosine and guanosine nucleotide pools but not into pyrimidine nucleotides. Thus, M. thermoautotrophicum can utilize exogenous adenosine by direct phosphorylation without hydrolysis of the glycosidic bond, and AMP can be efficiently converted to GMP. Factor F₀ and 7-methylpterin had approximately the same specific activities as the purine nucleotides. It follows that the ribityl side chain of factor F₀ is derived from the ribose side chain of a nucleotide precursor by reduction. The pyrazine ring of methanopterin is formed by ring expansion involving the ribose side chain of the precursor, GTP.Abbreviations Factor F₀ 8-hydroxy-6,7-didemethyl-5-deazariboflavin - APRT adenine phosphoribosyltransferase - GPRT guanine phosphoribosyltransferase - PRPP phosphoribosylpyrophosphate - HPLC high performance liquid chromatography     

Structure and function of hemoglobin in antarctic fishes and evolutionary implications

Summary The hematological features of cold-adapted, red-blooded Antarctic teleosts has prompted this study on the relationship between hemoglobin molecular structure and oxygen-binding properties. The hemolysates from 21 species of 5 families contained one component (Hb 1), often accompanied by an additional, minor one (Hb 2, 5%–10% of total). On the other hand, 3 species of Zoarcidae, a non-endemic family, had 4–5 components. All purified hemoglobins from the former group, but only 1–2 of the 4–5 hemoglobins of Zoarcidae, showed a strong Root effect (pH regulation of oxygen binding). Globins from each hemoglobin have been purified and characterised with respect to molecular structure in several species. The similarity between the complete amino acid sequence of one -chain and those of non-Antarctic -chains is lower than that among the latter sequences, suggesting independent pathways of evolution.Presented at the 5th SCAR Symposium on Antarctic Biology, Hobart, Australia (August 29th-September 3rd, 1988)     

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.     

Angiotensin converting enzyme inhibitor potentiated the vasorelaxant response of atrial natriuretic peptide in toad aortic rings

1. The vasorelaxant effect of synthetic atrial natriuretic peptide (ANP) on the vascular response to angiotensin II (A II) and norepinephrine (NE) in aortic rings from Bufo arenarum toad was studied. 2. Pretreatment with ANP partially inhibited the vascular response to A II and NE. 3. Angiotensin converting enzyme inhibitor (ACEI) treatment partially inhibited the contractile response of angiotensin I (A I) and did not affect the A II response. 4. The inhibitory effect of ANP on vascular response to A II and NE were potentiated by pretreatment with ACEI. 5. Results suggest that the angiotensin converting enzyme present in the vascular wall from Bufo arenarum toad may be involved in the metabolism of ANP.