Analyses of spatial patterns and population processes of clonal plants

The nonrandom spatial structure of terrestrial plants is formed by ecological interactions and reproduction with a limited dispersal range, and in turn this may strongly affect population dynamics and population genetics. The traditional method of modelling in population ecology is either to neglect spatial pattern (e.g. in transition matrix models) or to do straightforward computer simulation. We review here three analytical mothods to deal with plant populations in a lattice-structured habitat, which propagate both by seeds that scatter over the whole habitat and by vegetative reproduction (producing runners, rhizomes, etc.) to neighboring vacant sites. [1]Dynamics of global and local densities: Dynamical equations of population density considering nearest-neighbor correlation (spatial clumping) are developed as the joint dynamics of global average density and local density (comparable to mean crowding) based onpair approximation. If there is a linear trade-off between seed production and vegetative reproduction, the equilibrium abundance of the population may be maximized by engaging both means of reproduction. This result is accurately predicted by the pair approximation method, but not by mean-field approximation (neglect of spatial structure). [2]Cluster size distributions: Using global and local densities obtained by pair approximation, we predicted cluster size distribution, i.e. the number of clusters of occupied sites of various sizes. [3]Clonal identity probability decreasing with distance: Multi-locus measurement of allozymes or other neutral molecular markers tells us whether or not a given pair of individuals belong to the same clone. From the pattern of clonal identity probability decreasing with the distance between ramets, we can estimate the relative importance of two modes of reproduction: vegetative propagation and sexual seed production.     

Ligand recognition in μ opioid receptor: experimentally based modeling of μ opioid receptor binding sites and their testing by ligand docking

For three-dimensional understanding of the mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. As a first step, we have constructed molecular models for the multiple opioid receptor subtypes using bacteriorhodopsin as a template. The S-activated dihydromorphine derivatives should serve as powerful tools in mapping the three-dimensional structure of the μ opioid receptor, including the nature of the agonist-mediated conformational change that permits G protein-coupling to ‘second messenger’ effector molecules, and in identifying specific ligand-binding contacts with the μ opioid receptor. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments.     

Efficient production of anti-(hepatitis B virus) antibodies and their neutralizing activity in chimpanzees

 For industrial production of human monoclonal antibodies (hmAb) against hepatitis B virus surface antigen (HBsAg), we scaled-up a short-term perfusion culture in serum-free medium, which was chosen as the most suitable culture method, to a 50-l fermentor equipped with a rotating shear filter. Using hydrophobic chromatography as the initial step of hmAb purification, the mAb HBW4, HBW6 and W471 were isolated in good quality from the respective culture broths in yields of approximately 75%. Each of the three purified hmAb alone, and a cocktail of the three, protected chimpanzees against HB virus, when injected intravenously 3 h after viral challenge, as long as the serum antibody levels were significant. A pharmacokinetic study using cynomolgus monkeys demonstrated that the hmAb have a long plasma half-life and bioavailability of approximately 76% upon intramuscular injection in primates. Thus, anti-HBsAg hmAb produced by an industrial process are expected to be successfully used in clinical fields. Received: 20 June 1994/Received revision: 16 September 1994/Accepted: 10 October 1994     

A novel monoantennary complex-type sugar chain found in octopus rhodopsin: occurrence of the Gal{beta}1->4Fuc group linked to the proximal N-acetylglucosaniine residue of the trimannosyl core

The N-linked sugar chains were liberated as oligosaccha-ridesfrom octopus rhodopsin by hydrazinolysis. Most of the oligosaccharideswere neutral, and separated into two major components by columnchromatography using immobilized lectins and Bio-Gel P-4. Structuralanalysis of the one major component by sequential exoglycosidasedigestion, chemical fragmentation in combination with meth-ylationanalysis revealed that it is a nonasaccharide; Man16(Gaiβ13GlcNAcβ12Man13)Manβ14GlcNAcβ14(Galβ14Fuc16)GlcNAcThis structure is quite unique in that a novel galactosylatedfucose residue is attached to the reducing terminal N-acetyl-glucosamineresidue. galactosylated Fuc N-linked sugar chain novel structure octopus rhodopsin     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-l -cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [³H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na⁺-containing Tyrode's buffer, SNC-stimulated [³H]NA release was inhibited 30% by the coaddition of l -leucine. In the Na⁺-free, choline-containing buffer, SNC-stimulated [³H]NA release, which was similar to that in the Na⁺-containing buffer, was inhibited markedly by l -leucine, l -alanine, l -methionine, l -phenylalanine, and l -tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l -leucine was stronger than that of d -leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [³H]NA release in the Na⁺-free buffer. Uptake of l -[³H]leucine into the slices in the Na⁺-free buffer was inhibited by SNC, BCH, and l -phenylalanine, but not by l -lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l -leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na⁺-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Abnormally spliced messenger RNA in erythroid cells from patients with β+ thalassemia and monkey cells expressing a cloned β+-thalassemic gene

The reduced β-globin synthesis characterizing the β⁺ thalassemia phenotype has been shown to be caused by anomalous processing within the small Intervening sequence (IVS1) of the β-globin mRNA precursor. The β-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3′ junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3′-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with β⁺ thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned β⁺-thalassemic gene preferentially generated the anomalously spliced RNA when expressed In monkey kidney cells. The anomalously spliced RNA constituted approximately 80%–90%, and normal β RNA approximately 10%–20%, of the total β mRNA. In contrast, the small amount of β mRNA present in reticulocytes from such patients consisted predominantly of normal β mRNA. These results suggest that the reduced amount of normally functioning β mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent Instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.     

The selection of cultured plant cell lines producing high levels of biotin

We have found that biotin is synthesized in many species of cultured plant cells, e.g. Lavandula vera Labiatae), Nicotiana tabacum (Solanaceae) and Glycine max Leguminosae). Cultured green L. vera cells grown under light contained the greatest amounts of free biotin of the cells studied although the specific amounts varied among the cell lines. Cell lines were selected after their free biotin contents had been analysed. Cells containing large amounts of free biotin were cultured repeatedly, analysed and reselected. Lines with high levels of free biotin were obtained from cells which survived on a medium containing pimelic acid and l-alanine or from gamma irradiated cells. One L. vera cell line obtained from irradiated cells contained seven times the amount of free biotin found in the original unselected cultured cells and four and a half times that found in the leaves.