Non-lethal secondary product release from transformed root cultures of Beta vulgaris

Transformed root tissue of Beta vulgaris (Detroit Dark Red) was permeabilized to stimulate the release of intracellularly stored betanin without adverse affects on tissue viability as measured by biomass accumulation. Product release of up to 15% (w/w) was achieved by heat treatment at 42°C for 45 min with minimal effect on viability. Higher levels of product release were obtained with increasing temperature and exposure, but at the expense of viability. Viability was measured by comparing dry weight increases of permeabilized tissue 3 days after treatment vs non-permeabilized tissue over the same time interval. Recovery of heat-treated tissue was improved by addition of CaCl₂ (20 mm for 10 min) post-heat treatment. Betanin release up to 15% was also obtained at ambient temperature (25°C) by addition of up to 20 mm (NH₄)₂SO₄ in the presence of 1 mm ethylenediaminetetraacetic acid (EDTA). Correspondence to: A. A. DiIorio     

Future directions for root caries research

Interest among researchers in the diagnosis, aetiology, prevention, and treatment of root caries has increased substantially over the past two decades. However, there are some fundamental problems impeding the advancement of the field which remain to be addressed and resolved. A universally acceptable definition of root caries is not yet available. The relationship of root caries to coronal caries has not been established. The underlying disease process is still not clearly understood. The optimal utilisation of preventive/therapeutic agents for the treatment or prevention of root caries has not been determined. New treatment materials and preventive agents have not yet been tested in controlled clinical trials. These are a few of the issues and problems which we address in this paper.     

MOLECULAR TESTS OF THE HYPOTHESIZED HYBRID ORIGIN OF TWO DIPLOID HELIANTHUS SPECIES (ASTERACEAE)

Enzyme electrophoresis and restriction-fragment analysis of chloroplast DNA (cpDNA) and nuclear ribosomal DNA (rDNA) were used to test the hypothesis that both Helianthus neglectus and H. paradoxus are stabilized hybrid derivatives of H. annuus and H. petiolaris. The four species are annuals, diploid outcrossers, and have the same chromosome number. Helianthus annuus and H. petiolaris had the same allele in highest frequency for 16 of the 18 isozyme loci examined and had different majority alleles for only 6-Pgd3 and Pgi2. The two species had divergent rDNAs that could be distinguished by seven diagnostic restriction site mutations and three length mutations, and their cpDNAs could be differentiated by three diagnostic restriction site mutations. The alleles observed in H. neglectus were not a combination of those observed in H. annuus and H. petiolaris. Although H. neglectus had only one unique allele, it possessed none of the three alleles specific to H. annuus. In contrast, it had four of the seven alleles specific to H. petiolaris. Furthermore, H. neglectus had the same rDNA type as H. petiolaris and had the same cpDNA as that found in two populations of H. petiolaris ssp. fallax. These data allowed us to speculate that H. neglectus may be a recent derivative of H. petiolaris ssp. fallax, rather than a stabilized hybrid derivative as originally proposed. In contrast, H. paradoxus combined the alleles of H. annuus and H. petiolaris and had no unique alleles. At Adh2, H. paradoxus was monomorphic for an allele found only in H. petiolaris ssp. fallax, whereas at 6-Pgd3 and Pgi2, it was monomorphic for high frequency H. annuus alleles. Furthermore, H. paradoxus combined the rDNA repeat types of both proposed parents and had the chloroplast genome of H. annuus. These data provide compelling evidence that H. paradoxus, in contrast to H. neglectus, was derived via hybridization.     

Cloning of PCP1, a member of a family of pollen coat protein (PCP) genes from Brassica oleracea encoding novel cysteine-rich proteins involved in pollen-stigma interactions

The pollen coatings of both Brassica oleracea and Brassica napus contain a small family of basic 6–8 kDa proteins which are released on to the stigmatic surface on pollination. Following partial amino-acid sequencing of one of these pollen coat proteins (PCPs), PCR primers were constructed to isolate the PCP sequence from anther mRNA using RT-PCR. A cDNA was obtained which, in Northern hybridization experiments, revealed a characteristic pattern of expression during late stages of anther development. Interestingly, in situ hybridization revealed expression of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybridization experiments have shown the gene ( PCP1 ) to be a member of a large family of between 30 and 40 PCP genes in the genome of Brassica oleracea , Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F₂ segregants, perhaps resulting from the clustering of PCP sequences. PCP1 contains a single intron and encodes a small, basic peptide 83 amino acids in length featuring a hydrophobic signal peptide sequence separated from the more hydrophilic, cysteine-rich mature protein. The central part and C-terminal region of the peptide contain a characteristic and invariant pattern of eight cysteines which show clear homology with a number of other anther-specific genes; the remainder of the sequence shows little similarity to other sequences on the data bases. The product of PCP1 is a member of a large family of similar proteins, some of which have been demonstrated to bind specifically to S-locus glycoproteins, but does not appear to be genetically linked to the S-locus .     

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides

Porcine intestinal mucosal heparan sulfate was exhaustivelydepolymerized on a large scale using beparin lyase II (heparinaseII) or heparin lyase III (heparitinase, EC 4.2.2.8 [EC] ). The oligosaccharidemixtures formed with each enzyme were fractionated by low pressuregel permeation chromatography. Size-uniform mixtures of disaccharides,tetrasaccharides, and hexasaccharides were obtained. Each size-fractionatedmixture was then purified on the basis of charge by repetitivesemipreparative strong-anion-exchange high-performance liquidchromatography. This approach has led to the isolation of 13homogenous oligosaccharides. The purity of each oligosaccharidewas demonstrated by the presence of a single peak on analyticalstrong-anion-exchange high-performance liquid chromatographyand reversed polarity capillary electrophoresis. The structuresof these oligosaccharides were established using 500 MHz one-and two-dimensional nuclear magnetic resonance spectroscopy.Three of the thirteen structures that were solved were novelwhile the remaining 10 have been previously described. All ofthe structures obtained using heparin lyase III contained a     

Computer determination of perfusion patterns in pulmonary capillary networks

Hanger, Christopher C., Robert G. Presson, Jr., Osamu Okada,Steven J. Janke, John J. Watkins, Wiltz W. Wagner, Jr., and Ronald L. Capen. Computer determination of perfusion patterns in pulmonarycapillary networks. J. Appl. Physiol.82(4): 1283-1289, 1997.Individual pulmonary capillaries are notsteadily perfused. By using in vivo microscopy, it can readily bedemonstrated that perfusion continually switches between capillarysegments and between portions of the network within a single alveolarwall. These changes in capillary perfusion occur even when upstream pressure and flow are constant. Flow switching between capillary segments in the absence of hemodynamic changes in large upstream vessels suggests that capillary perfusion patterns could be random. Tocalculate the probability that perfusion patterns could occur bychance, it is necessary to know the total number of possible perfusionpatterns in a given capillary network. We developed a computer programthat can determine every possible perfusion pattern for any givencapillary network, and from that information we can calculate whetherperfusion of individual segments in the network is random. With theresults of the computer program, we have obtained statistical evidencethat some capillary segments in a network are nonrandomly perfused.

     

Sulfoxide configuration in sparsomycin determines time-dependent and competitive inhibition of peptidyl transferase

Sparsomycin, S_cR_s configuration, was the most potent of the four possible stereoisomers as a competitive inhibitor of peptide bond formation. In addition, the configuration of the two chiral centers dictated whether the compound exhibited time- and temperature-dependent inhibition of peptidyl transferase when incubated with polysomes prior to enzyme assay. The data corroborate the thesis that a peptidyl transferase-mediated acylation of the pivotal sulfoxide moiety and subsequent Pummerer rearrangement play a significant role in the inhibitory properties of sparsomycin.     

Two cDNA clones coding for the legumin protein of Pisum sativum L. contain sequence repeats

The sequence of two cDNA clones coding for the whole of the -subunit and most of the -subunit of legumin are presented together with a considerable amount of protein sequence data to confirm the predicted amino acid sequence. A unique feature shown by these cDNAs is the presence of three 56 base pair tandem repeats in the region encoding the C terminal of the polypeptide. The tandem repeats are also exhibited in the predicted polypeptide sequence as three 18 amino acid repeats which contain extremely high proportions of polar, mainly acidic, residues. The new sequences are compared to the previously published sequence of some shorter legumin cDNAs (Nature 295: 76–79). In the region where the sequences overlap, the previous cDNAs differ from the new ones by only a few base substitutions but most of the repeated region is not present though the sequences on either side are. The possibility that the absence of the repeats may reflect the difference between two types of legumin gene, rather than an artefact of the cloning of the cDNAs, is discussed.