Micropropagation ofPinus sylvestris

Plantlet regeneration from axillary buds, induced on seedling apices ofPinus sylvestris, is described. The influence of medium containing 1, 5, 10 or 50 M BA on the initiation of buds on 3, 6 and 9-week-old seedlings was investigated. With higher BA concentrations the number of apices that formed buds and the total number of buds increased, wheres their length decreased. Soaking the explants for 2 h in 111 M BA and for 5 h in 44 M BA gave better results, best expressed in longer buds. After 30 days, axillary buds were separated and transferred to a medium with 2% sucrose. Separated buds exposed to far-red light during the first week elongated better in comparison with the control. 64% of shoots rooted after a 24 h period of treatment with 53.8 M NAA incorporated in 0.6% water agar and transferred to 1/16-strength Murashige & Skoog medium as modified by Cheng supplemented with 1% sucrose.     

Immunological comparison of enzymes of the β-ketoadipate pathway

-Carboxy-cis,cis-muconate lactonizing enzyme and -carboxymuconolactone decarboxylase catalyze sequential reactions in the -ketoadipate pathway, the subunit sizes of the enzymes from Pseudomonas putida, biotype A, are 40000 and 13000, respectively. The cross reaction of antisera prepared against the enzymes was tested with the isofunctional enzymes formed by representatives of other bacterial species. Despite the differences in the subunit sizes of the enzymes, the antisera revealed the same general pattern: cross reaction was observed with the corresponding enzymes formed by other strains in the fluorescent Pseudomonas RNA homology group I and generally was not observed with enzymes from other Pseudomonas species or from other bacterial genera. Exceptions were provided by representatives of Pseudomonas cepacia. Members of this species are classified outside the fluorescent Pseudomonas RNA homology group. Nevertheless, the -carboxymuconolactone decarboxylases from these organisms formed precipitin bands with antisera prepared against the corresponding enzyme from P. putida, biotype A; the lactonizing enzymes from the two species did not appear to cross react. Immunodiffusion experiments with -carboxymuconolactone decarboxylase indicated that a common set of antigenic determinants for the enzyme is conserved among strains that have been classified together by other criteria; the relative immunological distances of the decarboxylases of each taxon from the reference P. putida, biotype A, enzyme were indicated by spurring patterns on Ouchterlony plates. These results suggested that the interspecific transfer of the structural gene for the enzyme is not a common event in Pseudomonas.Non-Standard Abbreviations CMLE -carboxy-cis,cis-muconate lactonizing enzyme (EC 5.5.1.2) - CMD -carboxymuconolactone decarboxylase (EC 4.1.1.44) - MLE cis,cis-muconate lactonizing enzyme (EC 5.5.1.1) - MI muconolactone isomerase (EC 5.3.3.4) Dedicated with affection and admiration to Professor R. Y. Stanier on his 60th birthday     

The effect of glycerol on the Chlorella symbiosis in Hydra viridis

In green hydra strains that are bleached by glycerol, photosynthesis is arrested in both intact hydra and freshly extracted algae whereas photosynthesis is not affected by glycerol in resistant hydra strains and their algae. Glycerol sensitivity is an inherent property of the algae and sensitivity can be transferred to resistant aposymbiotic hydra by infecting them with sensitive algae. It is suggested that the host hydra recognizes glycerol induced changes, other than photosynthetic incompetance, in the algae and either ejects or digests them.Australian Institute of Marine Science, P.M.B. No. 3, Townsville M.S.O. 4810 Australia     

Rooting apple cultivars in vitro: Interactions among light,temperature, phloroglucinol and auxin

Delicious apple (Malus domestica Borkh.) and several of its strains, which have been difficult to root in vitro, were successfully propagated with rooting percentages up to 100%. The combination of treatments used to achieve this result included placing the shoots on rooting medium in the dark at 30°C for the first week of the rooting stage, then moving them to a regime of 16 hr light-8 hr dark at 25°C. The rooting medium contained half strength Murashige and Skoog salts plus 1.2 M thiamine HCl, 0.56 mM myo-inositol, 1 mM phloroglucinol (PG), 1.4 M indolebutyric acid (IBA), 1.3 M gibberellic acid (GA₃), 87.6 mM sucrose, and 7 g l^–1 Difco Bacto agar. Dark treatment applied during the proliferation stage (etiolation) was less effective than one applied at the beginning of the rooting stage. The optimum length of dark treatment during rooting was 4 to 7 days. Increasing the temperature from 25°C to 30°C improved rooting of Delicious, Royal Red Delicious, and Vermont Spur Delicious in the absence of PG but generally had less effect in the presence of PG. Further increase in temperature to 35°C stimulated rooting of Royal Red Delicious but reduced rooting of Vermont Spur Delicious. Transfer of the cuttings to auxin-free medium after 1 week had no effect on percentage rooting and increased the number of roots per cutting for only 1 of 4 cultivars tested and then only in the presence of PG. In general PG stimulated rooting of Delicious and its strains, but had no effect on Golden Delicious.     

Heritable variation in the phaseolin protein of nondomesticated common bean,Phaseolus vulgaris L.

Summary Crude protein extracts from single seeds of nondomesticated Mexican bean accessions were analysed by SDS polyacrylamide gel electrophoresis for variability in phaseolin protein. Six new phaseolin types; M1, M2, M3, M4, M5, M6, which contained polypeptides within the same range of molecular weights (51,000 to 45,000 daltons) as occur in the S, T and C phaseolin types of cultivated beans were identified. No T and C types were found among the non-domesticated Mexican accessions, and the S type occurred in less than 7% of the seeds screened. Genetic analyses of F₂ progenies from crosses between Sanilac (S), and five of the M types showed that each M phaseolin phenotype was allelic to the S type and expressed codominantly.     

Induction of the Synthesis of Endo-1,4-β-xylanase and β-Galactosidase in Original and Recombinant Strains of the Fungus Penicillium canescens

The induction of synthesis of the secreted enzymes endo-1,4--xylanase (EC 3.2.1.8) and -galactosidase (EC 3.2.1.23) in original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for induction: the synthesis of -galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4--xylanase was observed at 5–10 mM. An increase in the number of endo-1,4--xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of -galactosidase; the synthesis of endo-1,4--xylanase in the high-copy-number recombinant producing -galactosidase was affected to a lesser extent. The amount of enzymes synthesized did not depend on the saccharide used as the sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Regulation of biosynthesis of aspartate family amino acids in the photosynthetic bacterium Rhodopseudomonas palustris

The four amino acids of the aspartate family (l-lysine, l-methionine, l-threonine, and l-isoleucine) are produced in bacteria by a branched biosynthetic pathway. Regulation of synthesis of early common intermediates and of carbon flow through distal branches of the pathway requires operation of a number of subtle feedback controls, which are integrated so as to ensure balanced synthesis of the several end products. Earlier studies with nonsulfur purple photosynthetic bacteria were instrumental in revealing the existence of alternative regulatory schemes, and in this communication we report on the control pattern of a representative of this physiological group not previously investigated, Rhodopseudomonas palustris. The results obtained from study of the properties of four key regulatory enzymes of the aspartate family pathway (-aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine deaminase) and of the effects of exogenous amino acids (i. e., the end products) on growth of the bacterium indicate that the control schema in Rps. palustris differs substantially from the schemes described for other Rhodopseudomonas species, but resembles the regulatory pattern observed in Rhodospirillum rubrum.Abbreviations A absorbancy - AK -aspartokinase - ASA aspartate -semialdehyde - DTT dithiothreitol - HS l-homoserine - HSDH homoserine dehydrogenase - HSK homoserine kinase - I l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-methionine - ME -mercaptoethanol - PABA p-aminobenzoic acid - T l-threonine - TD threonine deaminase - RCV synthetic growth medium (see text) - YP agar medium containing 0.3% yeast extract, 0.3% peptone, and 1.5% agar - Y2T synthetic growth medium (see text)     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

A simple method for maintaining high multiplication of Narcissus shoot cultures in vitro

A simple method for stimulating and maintaining high in vitro multiplication of Narcissus shoot clump cultures was developed. Shoot clumps were subjected either to normal cutting where leaves were trimmed to 20 mm in length at the beginning of each culture passage or to severe cutting where shoot clumps were cut down to the basal plate region removing all green tissue. Severe cutting at the beginning of each culture passage initially doubled the leaf multiplication, compared to normal cutting, but the difference between cutting treatments declined in successive passages. The improvement in leaf multiplication was maintained when shoot clumps were subjected to severe cutting only at every other culture passage, with no cutting in the alternate recovery passages. In vitro multiplication was increased by severe cutting in all seven Narcissus cultivars which were tested.Abbreviations NAA-1 naphthylacetic acid - BAP benzylaminopurine     

Glycoconjugates with Neu5Ac(α2,6)Gal(β1,4)GlcNAc sequences: a selective lectin-histochemical property of Kurloff cells in guinea pig thymus

Summary The Kurloff cell (KC), a natural killer lymphocyte, contains a large (10-m diameter) periodic acid-Schiff (PAS)-positive lysosome-like inclusion body called the Kurloff body (KB), which exhibits strong acid phosphatase activity. The presence of Sambucus nigra agglutinin (SNA)-reactive Neu5Ac(2,6)-D-Gal/Gal-NAc(1,4)GlcNAc oligosaccharide sequences and the absence of the corresponding Neu5Ac(2,3) Maackia amurensis agglutinin (MAA)-reactive sequence in the major 35-kDa N-glycosylproteins of the complex or hybrid type extracted from purified KC were established by Western-lectin-blotting of cytosolic extracts from purified KC. Moreover, these SNA-reactive sequences, or at least part of them, were shown to be borne by sialidase-sensitive KC acid isophosphatases. Thymic sections rich in KC, from estrogenized guinea pigs were examined by affino-histochemistry with these sialic acid-reactive lectins. The SNA-reactivity of thymic sections was quasiexclusively confined to KC clusters, whereas the whole thymic section was negative for MAA. KC were not SNA-reactive following preincubation and incubation with 200 mM lactose. When submitted to enzymatic or mild chemical desialylation processes, the SNA-reactivity of the KC clusters was enhanced. The SNA-reactivity of KC clusters was completely abolished following prolonged chemical desialylation, whereas the PAS-positivity of KB remained unchanged. Even after a prolonged sialidase treatment, this SNA-reactivity was only reduced. Moreover, after both these desialylation processes, KC developed a heavier Ricinus communis agglutinin-reactivity, thus confirming the presence of penultimate Gal residues in their abundant SNA-reactive oligosaccharide sequences Neu5Ac(2,6)Gal(1,4)GlcNAc. Such a selective lectin histochemical property provides a marker for detecting KC.     

Early development of the self-fertilizing mangrove killifish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> reared in the laboratory

The mangrove killifish Rivulus marmoratus was reared at 25°±1°C and 17ppt salinity from 0 to 100 days after hatching (DAH), and its early development was described by examining growth and morphometric parameters, meristic characters (vertebral and fin-ray counts), bone-cartilage development, and pigmentation. Growth was isometric for preanal length, head length, snout length, body depth, pectoral-fin length, dorsal-fin length, anal-fin length, and caudal-peduncle depth. Negative allometric growth was observed in eye diameter and gape size. Meristic counts (mean±SD) for vertebrae (34.2±0.4) and dorsal- (8.6±0.5), anal- (11.4±0.5), and caudal-fin rays (30.2±0.8) were complete at 0 DAH (n=5), whereas pectoral-fin rays and pelvic-fin rays were complete by 30 DAH (14.5±0.4, n=5) and 60 DAH (4.2±0.8, n=5). Full ossification of meristic elements proceeded in the following sequence: vertebrae (by 30 DAH), caudal-, dorsal-, and anal-fin rays (by 60 DAH), pectoral-fin rays (between 60 DAH and 100 DAH), and pelvic-fin rays (by 100 DAH). Both morphological characters and meristic counts indicate that this species can be considered to be a juvenile after 9.8mm in standard length (20 DAH).     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.     

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: 3)--L-QuipNAc-(13)--D-GlcpNAc-(16)--D-GlcpNAc-(1 (S)-Lac-(2–3) (1) 4)--D-GlcpA-(13)--D-GalpNAc-(14)--D-Glcp-(13)--D-Galp-(14)--D-GalpNAc-(1 L-Ala-(2–6) (2) 3)--D-GalpNAc-(16)--D-GalpNAc-(14)--D-GlcpA-(1 L-Lys-(2–6)--D-GalpA-(14) (3) 4)--D-GlcpA-(16)--D-GalpNAc-(16)--D-GlcpNAc-(13)--D-GlcpNAc-(1 (R)-aLys-(2–6) (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N-[(R)-1-carboxyethyl]-L-lysine (alaninolysine), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Polymerization Sites in the D-Domain of Human Fibrin(ogen)

The present work deals with localization of previously unknown polymerization sites of the fibrin DD-fragment. D-dimer we obtained has a pronounced inhibitory effect on fibrin polymerization (IC₅₀ = 0.06 M). The inhibitory effect of the D-fragment disappeared after reduction and carboxymethylation. However, polypeptide chains _DD (B134-461) and _DD (63-411)₂ of the DD-fragment, isolated by preparative electrophoresis, displayed their inhibitory activity. For instance, the rates of fibrin protofibril lateral association were decreased twice in the presence of _DD and _DD chains at their molar ratios to fibrin of 0.40 and 0.15, respectively. The IC₅₀ values for _DD and _DD were 0.24 and 0.10 M, respectively. Highly specific inhibition of protofibril lateral association suggests that the protofibril lateral association sites are located in B134-461 and 63-411 regions of the fibrin D-domain. Our data confirm those reported by Doolittle et al. regarding the -chain and a hypothesis about -chain of fibrin D-domain (Yang, Z., Mochalkin, I., and Doolittle, R. F. (2000) Biochemistry, 97, 14156-14161).     

Inheritance and linkage of isozyme loci in almond

The segregation of seven isozyme marker genes was investigated using eight controlled crosses in almond. The cultivar Nonpareil was the maternal parent in all crosses. Pollination was achieved using eight different cultivars, and a total of 3200 individual kernels were assessed. For each isozyme the goodness-of-fit test was used to test for departure from the expected frequencies assuming Mendelian inheritance. Given a higher than expected number of significant results for individual isozymes, independent segregation between pairs of isozymes was tested using the chi-square statistic on the resulting two-way contingency tables. In all crosses a highly significant association (P value< 0.001) was observed between (1) the AAT- 1 and IDH isozymes loci and (2) the LAP-1 and PGM-2 isozymes loci, which leads to the conclusion that the respective isozyme pairs are linked.In addition, a significant association (P value < 0.001) was observed between LAP-1 and GPI-2 when the pollen sources were Fritz, Mission, or Price, but this could not be tested for the remaining five pollen sources, Carmel, Grant, Keane, Ne plus Ultra, Peerless, because they are homozygous at these loci. If LAP-1 is linked with GPI-2 and PGM-2, it might be expected that we should find evidence of linkage between GPI-2 and PGM-2. The lack of a significant association between these two isozymes suggests that LAP-1 is located centrally on the chromosome. These three pairs of linked loci are the first to be reported in almond.