IDENTIFICATION OF CYLINDROSPERMOPSIN IN APHANIZOMENON OVALISPORUM (CYANOPHYCEAE) ISOLATED FROM LAKE KINNERET,ISRAEL1

Aphanizomenon ovalisporum (Forti) was identified and isolated from Lake Kinneret upon its first appearance as a dominant bloom in late 1994. This cyanobacterial species, not previously known to be toxic, was evaluated by a commonly used mouse bioassay and was demonstrated to induce toxic symptoms that were distinguishable from the typical symptoms of the neurotoxins previously reported in Aphanizomenon flos-aquae (L.) Ralfs. Mice died 5–24 h after crude extracts were injected intraperitoneally, and the LD₅₀ value was estimated as 465 mg dry wt biomass · kg^?1 mouse. A toxicity-guided fractionation of the active extract indicated that the potent substance is polar an nature. The structure of the active compound was determined by its mass spectrometry and NMR data. The compound was found to be the sulfate-guanidinium zwitterion, cylindrospermopsin, previously isolated from the cyanobacterium Cylindrospermopsis raciborskii (Woloszynska) and recently also reported in Umezakia natans (Watanabe). This is the first time that Aphanizomenon ovalisporum has been reported to contain a toxic compound.     

Analysis of the steady state binding, internalization, and degradation of human interferon-alpha2

Scatchard analyses of the equilibrium binding of radiolabeled human interferon-alpha2 (huIFN-alpha2) to Madin-Darby bovine kidney cells previously exposed to subsaturating concentrations of IFN-alpha showed approximately a 50% decrease in the number of cell surface receptors and no change in the apparent dissociation constant, Kd, compared with cells not exposed to interferon. The steady state equations describing the interaction of polypeptide ligands with cell surface receptors under physiological conditions (Wiley, H.S., and Cunningham, D.D. (1981) Cell 25, 433-440) have allowed us to determine, under steady state conditions, the rate of insertion of receptors into the cell membrane, the endocytic rate constant of occupied receptors, the rate constant of turnover of unoccupied receptors, and the rate of hydrolysis of internalized ligand. Our results indicate that occupied and unoccupied interferon receptors are cleared from the cell surface at approximately the same rate. This suggests that the down-regulation of the huIFN-alpha2 receptor on Madin-Darby bovine kidney cells by huIFN-alpha2 differs from that of several other surface receptors for polypeptide hormones and growth factors analyzed on cultured cells in that the binding of huIFN-alpha2 to its receptor does not increase the rate of receptor endocytosis.     

PCR-generated molecular markers for the invertase gene and sucrose accumulation in tomato

The green-fruited tomato species, Lycopersicon hirsutum, unlike the domesticated red-fruited species, L. esculentum, accumulates sucrose during the final stages of fruit development, concomitant with the loss of soluble acid invertase activity. In order to study the genetic linkage of sucrose accumulation to the invertase gene, part of the invertase gene from L. hirsutum was cloned, sequenced and the sequence compared with the invertase sequence of the red-fruited L. esculentum. Several base changes were found in the coding region of the two invertase genes. Based on these base -pair differences, we developed a species-specific PCR assay capable of determining, in a single PCR reaction, the origin of the invertase gene in segregating seedlings of an interspecific cross. Our results indicate that the invertase gene is genetically linked to sucrose accumulation in the green-fruited L. hirsutum.     

The Glycine Site of the N-Methyl-D-Aspartate Receptor Channel: Differences Between the Binding of HA-966 and of 7-Chlorokynurenic Acid

The mechanisms of action of three different glycine-site antagonists of the N-methyl-D-aspartate (NMDA)-receptor channel were analyzed employing [3H]glycine direct binding assays, as well as functional glycine- and glutamate-induced uncompetitive blocker binding assays. The latter assays measure apparent channel opening. All three antagonists tested, viz., 7-chlorokynurenic acid (7-Cl-KYNA), kynurenic acid (KYNA), and 1-hydroxy-3-aminopyrrolidone-2 (HA-966), inhibited the binding of [3H]glycine to the NMDA receptor in a dose-dependent manner. These antagonists also inhibited the glycine-induced increase in accessibility of the uncompetitive blocker [3H]N-[1-(2-thienyl)cyclohexyl]-piperidine ([3H]TCP) to the channel. 7-Cl-KYNA and KYNA, but not HA-966, completely blocked the glutamate-induced binding of [3H]TCP, in a manner similar to the non-competitive manner in which the selective NMDA antagonist D-(-)-2-amino-5-phosphonovaleric acid (AP-5) inhibited glycine-induced [3H]TCP binding. The inhibitory effects of HA-966 and of AP-5 on glutamate-induced [3H]TCP binding were overcome when glutamate concentrations were increased. Of the three antagonists, 7-Cl-KYNA appears to be the most potent (Ki = 0.4-1.0 microM) and the most selective glycine antagonist. KYNA was found to act at both the glycine (Ki = 40-50 microM) and the glutamate sites. In contrast, HA-966 (Ki = 6-17 microM) appears to act either on a domain distinct from the glutamate and the glycine sites, but tightly associated with the latter, or at the glycine site, but according to a mechanism distinct from that of 7-Cl-KYNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Host defence versus intraspecific competition in the regulation of infrapopulations of the flea Xenopsylla conformis on its rodent host Meriones crassus

Mechanisms that regulate parasite populations may influence the evolution of hosts and parasites, as well as the stability of host-parasite dynamics but are still poorly understood. A manipulation experiment on the grooming ability of rodent hosts (Meriones crassus) and flea (Xenopsylla conformis) densities on these hosts successfully disentangled two possible regulating mechanisms: (i) behavioural defence of the host and (ii) intraspecific competition among parasites, and revealed their importance in suppressing the feeding of fleas. Moreover, the results suggest that flea competition is direct and is not mediated by host grooming, immune response, or parasite-induced damage to the host. These mechanisms, together with interspecific competition and density-dependent parasite-induced host damage, may limit the parasite burden on an individual host and may prevent parasites from overexploiting their host population.     

The expression pattern and cellular localisation of Myosin VI during the Drosophila melanogaster life cycle

Myosin VI is a motor protein which is necessary for the morphogenesis of epithelial tissues during Drosophila development. The spatial and temporal expression of Myosin VI was examined by expressing a GFP (Green Fluorescent Protein) tagged Myosin VI molecule (PGM), under the control of a Myosin VI-Gal4 line. PGM was present in tissues that were shown previously to express Myosin VI, such as the ovarian follicle epithelium, and the individualization complex; and in other tissues, including the trachea, the midgut, the salivary glands and the imaginal discs. The GFP-tagged Myosin V1 rescued the male sterile phenotype of Jaguar showing it is functional in vivo. Within individual cells, the role of the head and neck domain and the tail domain in targeting of the Myosin V1 molecule was examined by investigating the localisation of the separate domains tagged to GFP. In salivary glands and follicle cells the head and neck domains were concentrated in the cell nucleus, where the minus end of each actin filament is located. We found that the tail domain anchors the whole molecule outside of the nucleus. Similarly, in the individualization complex in the testes, the tail anchors the whole molecule to the base of the complex while the separated head with neck domain becomes scattered along the entire actin molecule suggesting the cellular location may be determined by cargo proteins that bind to the tail domain rather than by the movement of Myosin VI along the actin filaments.     

Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus.

The RNA-stimulated nucleoside triphosphatase (NTPase) and helicase of hepatitis C virus (HCV) consists of three domains with highly conserved NTP binding motifs located in the first domain. The ATP-binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and it was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364 of the HCV polyprotein, was confirmed by N- and C-terminal sequencing and by its capability to bind 5'-fluorosulfonylbenzoyladenosine (FSBA). The analyses of the kinetics of ATP binding revealed a single class of binding site with the Kd of 43.6 microM. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as regulators of the ATP-binding capacity of the domain. Polynucleotides bind to the domain and enhance its affinity for ATP. In addition, ATP enhances the affinity of the domain for the polynucleotides. Different compounds, which are known to interact with nucleotide binding sites of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents behaved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50 = 22 microM), and trifluoperazine, which inhibits the ATP binding by a noncompetitive mechanism (IC50 = 98 microM). Kinetic experiments with the NTPase/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP-binding domain.     

Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO(2) limitation and oxidative stress.

The phytoplankton assemblage in Lake Kinneret is dominated in spring by a bloom of the dinoflagellate Peridinium gatunense, which terminates sharply in summer [1]. The pH in Peridinium patches rises during the bloom to values higher than pH9 [2] and results in CO(2) limitation. Here we show that depletion of dissolved CO(2) (CO(2(dis))) stimulated formation of reactive oxygen species (ROS) and induced cell death in both natural and cultured Peridinium populations. In contrast, addition of CO(2) prevented ROS formation. Catalase inhibited cell death in culture, implicating hydrogen peroxide (H(2)O(2)) as the active ROS. Cell death was also blocked by a cysteine protease inhibitor, E-64, a treatment which stimulated cyst formation. Intracellular ROS accumulation induced protoplast shrinkage and DNA fragmentation prior to cell death. We propose that CO(2) limitation resulted in the generation of ROS to a level that induced programmed cell death, which resembles apoptosis in animal and plant cells. Our results also indicate that cysteine protease(s) are involved in processes that determine whether a cell is destined to die or to form a cyst.