Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

1,8-Cineole inhibits root growth and DNA synthesis in the root apical meristem ofBrassica campestris L.

1,8-cineole is a volatile growth inhibitor produced bySalvia species. We examined the effect of this allelopathic compound on the growth of other plants usingBrassica campestris as the test plant. Cineole inhibited germination and growth ofB. campestris in a dosedependent manner. WhenB. campestris was grown for 5 days with various concentrations of cineole, the length of the roots was found to be shorter as the concentration of cineole increased, whereas the length of the hypocotyl remained constant up to 400 μM cineole, indicating that cineole specifically inhibited growth of the root. The mitotic index in the root apical meristem of 3-day-old seedlings decreased from 5.6% to 1.6% when exposed to 400 μM cineole, showing that cineole inhibits the proliferation of root cells. We then examined the effect of cineole on DNA synthesis by indirect immunofluorescence microscopy using antibody raised against 5-bromo-2′-deoxyuridine (BrdU, an analogue of thymidine) in thin sections of samples embedded in Technovit 7100 resin. The results clearly demonstrated that cineole inhibits DNA synthesis in both cell nuclei and organelles in root apical meristem, suggesting that cineole may interfere with the growth of other plant species by inhibiting DNA synthesis in the root apical meristem.     

The effects of calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases

The polyhydroxylated nortropane alkaloids called calyste-ginesoccur in many plants of the Convolvulaceae, Solanaceae, andMoraceae families. Certain of these alkaloids exhibit potentinhibitory activities against glycosidases and the recentlydemonstrated occurrence of calystegines in the leaves, skins,and sprouts of potatoes (Solatium tuberosum), and in the leavesof the eggplant (S.melongena), has raised concerns regardingthe safety of these vegetables in the human diet. We have surveyedthe occurrence of calystegines in edible fruits and vegetablesof the families Convolvulaceae, Solanaceae, and Moraceae byGC-MS. Calystegines A3, B1, B2, and C1 were detected in allthe edible fruits and vegetables tested; sweet and chili peppers,potatoes, eggplants, tomatoes, Physalis fruits, sweet potatoes,and mulberries. Calystegines B1 and C1 were potent competitiveinhibitors of the bovine, human, and rat β-glucosidaseactivities, with K₁ values of 150, 10, and 1.9 µM, respectivelyfor B1 and 15,1.5, and 1 µM, respectively, for C1. CalystegineB2 was a strong competitive inhibitor of the -galactosidaseactivity in all the livers. Human β-xylosidase was inhibitedby all four nortropanes, with calystegine C1 having a K₁ of0.13 µM. Calystegines A3 and B2 selectively inhibitedthe rat liver β-glucosidase activity. The potent inhibitionof mammalian β-glucosidase and -galactosidase activitiesin vitro raises the possibility of toxicity in humans consuminglarge amounts of plants that contain these compounds. edible plants calystegines glycosidase inhibitors bovine, human, and rat liver     

Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis

We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl₂ and MnCl_{_2}.     

Preliminary investigation of feeding performance of larvae of early red-spotted grouper, Epinephelus coioides, reared with mixed zooplankton

Larvae of red-spotted grouper, Epinephelus coioides, were reared inoutdoor tanks with nauplii of copepods (mainly Pseudodiaptomus annandaleiand Acartia tsuensis) and/or rotifers, Brachionus rotundiformis. Grouperlarvae successfully started feeding on early stage nauplii even though theirabundance was as low as approximately 100 individuals l^–1 andshowed better survival and growth thereafter compared to those fed withrotifers only. Incidence of feeding reached 100% on day 4 whennauplii were available and only on day 9 when rotifers were given alone.Larvae seemed to be poor feeders at the onset of feeding, attempting tocapture any food organisms in the tank water. Selective feeding ability oflarvae started from day 4 and the larvae then preferred to feed on medium-and large-size nauplii rather than on rotifers as they grew. Larvae appearedto have a better chance at surviving in the presence of early stage nauplii,which were probably caught more easily than rotifers.     

Interspecific interactions in the marine rotifer microcosm

Copepods and protozoans often co-exist in marinerotifer mass cultures. Interspecific interactionbetween the rotifer Brachionus rotundiformisTschugunoff and eight other zooplankton species,namely Brachionus plicatilis O. F. Müller (rotifer),Diaphanosoma celebensis Stingelin (cladoceran),Tigriopus japonicus Mori, Acartia sp. (copepod),Euplotes sp., Vorticella sp., an unidentifiedprotozoan species (P1 strain) (protozoan) and Artemiasp. (anostracan) at two developmental stages (nauplii –0.95 mm, 0 days old; adults – 3.3 mm, 19 days old) wereinvestigated in the laboratory.There was no contaminating species that contributedto an increase in rotifer population growth during theexperiments. Four types of interspecific interactionswere seen between B. rotundiformis and otherco-existing zooplankton species. These include effectson population growth: (1) both species declined, (2) onespecies is promoted while the other is not influenced,(3) one species is declined while the other is notinfluenced and (4) one species is promoted while theother declined. The first type was exhibited by B. rotundiformis vs B. plicatilis, B. rotundiformis vsD. celebensis and B. rotundiformis vs Artemia sp. The second type was exhibited by B. rotundiformis vsVorticella sp. and the third type by B. rotundiformisvs Euplotes sp. and B. rotundiformis vs T. japonicus. The fourth type was exhibited by B. rotundiformis vs Acartia sp. and B. rotundiformis vs P1 strain.     

Specific interaction of polypyrimidine tract-binding protein with the extreme 3'-terminal structure of the hepatitis C virus genome, the 3'X.

We previously identified a highly conserved 98-nucleotide (nt) sequence, the 3'X, as the extreme 3'-terminal structure of the hepatitis C virus (HCV) genome (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215:744-749, 1995). Since the 3' end of positive-strand viral RNA is the initiation site of RNA replication, the 3'X should contribute to HCV negative-strand RNA synthesis. Cellular factors may also be involved in this replication mechanism, since several cellular proteins have been shown to interact with the 3'-end regions of other viral genomes. In this study, we found that both 38- and 57-kDa proteins in the human hepatocyte line PH5CH bound specifically to the 3'-end structure of HCV positive-strand RNA by a UV-induced cross-linking assay. The 57-kDa protein (p57), which had higher affinities to RNA probes, recognized a 26-nt sequence including the 5'-terminal 19 nt of the 3'X and 7 flanking nt, designated the transitional region. This sequence contains pyrimidine-rich motifs and shows similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), which has been implicated in alternative pre-mRNA splicing and cap-independent translation. We found that this 3'X-binding p57 is identical to PTB. The 3'X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB bound to the 3'X RNA. In addition, p57 bound solely to the 3'-end region of positive-strand RNA, not to this region of negative-strand RNA. We suggest that 3'X-PTB interaction is involved in the specific initiation of HCV genome replication.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.