Changes in Morphology and Elemental Composition of <Emphasis Type="Italic">Vibrio splendidus</Emphasis> Along a Gradient from Carbon-limited to Phosphate-limited Growth

We examined morphology, elemental composition (C, N, P), and orthophosphate-uptake efficiency in the marine heterotrophic bacterium Vibrio splendidus grown in continuous cultures. Eight chemostats were arranged along a gradient of increasing glucose concentrations in the reservoirs, shifting the limiting factor from glucose to phosphate. The content of carbon, nitrogen, and phosphorus was measured in individual cells by x-ray microanalysis using a transmission electron microscope (TEM). Cell volumes (V) were estimated from length and width measurements of unfixed, air-dried cells in TEM. There was a transition from coccoid cells in C-limited cultures toward rod-shaped cells in P-limited cultures. Cells in P-limited cultures with free glucose in the media were significantly larger than cells in glucose-depleted cultures (P < 0.0001). We found functional allometry between cellular C-, N-, and P content (in femtograms) and V (in cubic micrometers) in V. splendidus (C = 224 × V ^0.89, N = 52.5 × V ^0.80, P = 2 × V ^0.65); i.e., larger bacteria had less elemental C, N, and P per V than smaller cells, and also less P relative to C. Biomass-specific affinity for orthophosphate uptake in large P-limited V. splendidus approached theoretical maxima predicted for uptake limited by molecular diffusion toward the cells. Comparing these theoretical values to respective values for the smaller, coccoid, C-limited V. splendidus indicated, contrary to the traditional view, that large size did not represent a trade-off when competing for the non-C-limiting nutrients.     

Anticonvulsant Efficacy of Drugs with Cholinergic and/or Glutamatergic Antagonism Microinfused into Area Tempestas of Rats Exposed to Soman

A group of antiparkinson drugs (benactyzine, biperiden, caramiphen, procyclidine, and trihexyphenidyl) has been shown to possess both anticholinergic and antiglutamatergic properties, making these agents very well suited as anticonvulsants against nerve agents. The first purpose of this study was to make a comparative assessment of the anticonvulsant potencies of the antiparkinson agents when microinfused (1 μl) into the seizure controlling area tempestas (AT) of rats 20 min before subcutaneous injection of soman (100 μg/kg). The second purpose was to determine whether cholinergic and/or glutamatergic antagonism was the effective property. The results showed that only procyclidine (6 μg) and caramiphen (10 μg) antagonized soman-induced seizures. Cholinergic, and not glutamatergic, antagonism was likely the active property, since atropine (100 μg), and scopolamine (1 μg) caused anticonvulsant effects, whereas MK-801 (1 μg), and ketamine (50 μg) did not. Soman (11 nmol) injected into AT resulted more frequently in clonic convulsions than full tonic–clonic convulsions. AT may serve as both a trigger site for soman-evoked seizures and a site for screening anticonvulsant potencies of future countermeasures. Special issue article in honor of Dr. Frode Fonnum.     

Titimbera,a new genus of Orthocladiinae from South and Central America (Diptera: Chironomidae)

Titimbera n. gen. is erected based on the males of three new species from South and Central America: T. amazonica n. sp. from the Amazon region, Brazil; T. titi n. sp. from Venezuela and T. laselvensis n. sp. from Costa Rica. The combination of bare eyes and wing membrane; antenna without strong apical seta; scalpellate acrostichals in mid scutum; costa strongly extended; R₄₊₅ ending opposite to M₃₊₄; Cu₁ strongly curved to slightly sinuous; anal point sitting high on tergite IX, nearly parallel-sided with bluntly rounded apex; and club-shaped to subtriangular gonostylus with distinct heel will separate the genus from all other orthoclads.     

Hypoxia-Induced Apoptosis in Human Cells with Normal p53 Status and Function, without Any Alteration in the Nuclear Protein Level

We have studied hypoxia-induced inactivation of cells from three established human cell lines with different p53 status. Hypoxia was found to induce apoptosis in cells expressing wild-type p53 (MCF-7 cells), but not in cells where p53 is either mutated (T-47D cells), or abrogated by expression of the HPV18 E6 oncoprotein (NHIK 3025 cells). Apoptosis was demonstrated by DNA fragmentation, using agarose gel electrophoresis of DNA and DNA nick end labeling (TUNEL). We demonstrate that extremely hypoxic conditions (<4 ppm O₂) do not cause any change of expression in the p53 protein level in these three cell lines. In addition, the localization of p53 in MCF-7 cells was found exclusively in the nucleus in only some of the cells both under aerobic and hypoxic conditions. Furthermore, no correlation was found between the p53-expression level and whether or not a cell underwent apoptosis. Flow cytometric TUNEL analysis of MCF-7 cells revealed that initiation of apoptosis occurred in all phases of the cell cycle, although predominantly for cells in S phase. Apoptosis was observed only during a limited time window (i.e., ≈10 to ≈24 h) after the onset of extreme hypoxia. While 66% of the MCF-7 cells lost their ability to form visible colonies following 15 h exposure to extreme hypoxia, only ∼28% were induced to apoptosis, suggesting that ∼38% were inactivated by other death processes. Commitment to apoptotic cell death was observed in MCF-7 cells even for oxygen concentrations as high as 5000 ppm. Our present results indicate that the p53 status in these three tumor cell lines does not have any major influence on cell's survival following exposure to extremely hypoxic conditions, whereas following moderate hypoxia, cells expressing functional p53 enhanced their susceptibility to cell death. Taken together, although these results suggest that functional p53 might play a role in the induction of apoptosis during hypoxia, other factors seem to be equally important.     

Properties and action pattern of the recombinant mannuronan C-5-epimerase AlgE2

The mannuronan C-5-epimerase AlgE2 is one of a family of Ca²⁺-dependent epimerases secreted by Azotobacter vinelandii. These enzymes catalyze the conversion of β- -mannuronic acid residues (M) to - -guluronic acid residues (G) in alginate. AlgE2 has been produced by fermentation with a recombinant strain of Escherichia coli, isolated and partially purified. Epimerization with AlgE2 increased the content of G-residues in different alginates from starting values of 0–45% up to approximately 70%. The new G-residues were mainly present in short blocks. Although G-residues may be introduced next to pre-existing G-residues, AlgE2 was not able to epimerize strictly alternating MG-structures. The epimerization with AlgE2 was greatly affected by the concentration of Ca²⁺. The type of alginate used as substrate affected the reaction rate and the reaction pattern especially at low Ca²⁺ concentration. AlgE2 appears to act by a preferred attack mechanism where the enzyme associates with different sequences in the alginate depending on the concentration of Ca²⁺. During epimerization, AlgE2 occasionally causes cleavage of the alginate chain. The observed frequency corresponds to 1–3 breaks per 1,000 M-units epimerized.     

1,5-Disubstituted 1,2,3-triazoles as cis-restricted analogues of combretastatin A-4: Synthesis, molecular modeling and evaluation as cytotoxic agents and inhibitors of tubulin

A series of cis-restricted 1,5-disubstituted 1,2,3-triazole analogues of combretastatin A-4 (1) have been prepared. The triazole 12f, 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)aniline, displayed potent cytotoxic activity against several cancer cell lines with IC₅₀ values in the nanomolar range. The ability of triazoles to inhibit tubulin polymerization has been evaluated, and 12f inhibited tubulin polymerization with IC₅₀ = 4.8 μM. Molecular modeling experiments involving 12f and the colchicine binding site of ,β-tubulin showed that the triazole moiety interacts with β-tubulin via hydrogen bonding with several amino acids.     

Candicidin Biosynthesis Gene Cluster Is Widely Distributed among Streptomyces spp. Isolated from the Sediments and the Neuston Layer of the Trondheim Fjord,Norway

A large number of Streptomyces bacteria with antifungal activity isolated from samples collected in the Trondheim fjord (Norway) were found to produce polyene compounds. Investigation of polyene-containing extracts revealed that most of the isolates produced the same compound, which had an atomic mass and UV spectrum corresponding to those of candicidin D. The morphological diversity of these isolates prompted us to speculate about the involvement of a mobile genetic element in dissemination of the candicidin biosynthesis gene cluster (can). Eight candicidin-producing isolates were analyzed by performing a 16S rRNA gene-based taxonomic analysis, pulsed-field gel electrophoresis, PCR, and Southern blot hybridization with can-specific probes. These analyses revealed that most of the isolates were related, although they were morphologically diverse, and that all of them contained can genes. The majority of the isolates studied contained large plasmids, and two can-specific probes hybridized to a 250-kb plasmid in one isolate. Incubation of the latter isolate at a high temperature resulted in loss of the can genes and candicidin production, while mating of the “cured” strain with a plasmid-containing donor restored candicidin production. The latter result suggested that the 250-kb plasmid contains the complete can gene cluster and could be responsible for conjugative transfer of this cluster to other streptomycetes.Actinomycete bacteria, especially those belonging to the family Streptomycetaceae, are well-known producers of secondary metabolites with diverse biological activities. Representatives of the genus Streptomyces produce a variety of antibiotics with antibacterial, antifungal, and antitumor activities. The majority of antibiotic-producing streptomycetes have been isolated from terrestrial environments, while antibiotic-producing streptomycetes from the marine sources remain largely unexplored. Therefore, studies of streptomycetes from the marine environment are important for unraveling their potential for antibiotic production. In addition, such studies might reveal the means by which antibiotic biosynthesis and resistance genes are spread in nature.It is widely acknowledged that plasmids play an important role in genetic exchange between bacterial species. Conjugative plasmids are quite common in Streptomyces strains (13), and a number of these mobile genetic elements have been characterized in detail. The characterized mobile genetic elements include both circular plasmids, such as pIJ101 from Streptomyces lividans (14) and SCP2 from Streptomyces coelicolor (2, 35), and linear plasmids, such as SLP2 from S. lividans (6) and SCP1 from S. coelicolor (38, 39). The presence of a linear plasmid in Streptomyces was first reported in 1979, and the plasmid was the 17-kb pSLA2 plasmid of Streptomyces rochei (11). SCP1 of S. coelicolor was discovered in the early 1970s (38, 39), but because of its large size (356 kb), isolation of this plasmid with conventional techniques was not possible and therefore it was not recognized as a linear plasmid until pulsed-field gel electrophoresis (PFGE) was invented. Later, SCP1 was shown to harbor a complete set of genes for biosynthesis of the antibiotic methylenomycin (21; K. F. Chater, C. J. Bruton, S. J. O''Rouke, and A. W. Wietzorrek, 5 July 2001, Patent Cooperation Treaty international application WO/2001/048228), while another linear plasmid, found in S. rochei, has been shown to contain genes for biosynthesis of both lankamycin and lankacidin (16, 19, 28, 36). Other examples of plasmids include pPZG103 carrying oxytetracycline biosynthesis genes acquired from the chromosome of Streptomyces rimosus (10) and pKSL from Streptomyces lasaliensis, which might be involved in the production of lasalocid and/or echinomycin (17, 20).Linear plasmids can be transferred between Streptomyces strains by means of conjugation, and SCP1 is an example of a conjugative linear plasmid as it is easily transferred from an SCP1⁺ strain to an SCP1⁻ strain (39). Interspecific transfer to S. lividans and Streptomyces parvulus has also been reported for this plasmid, and it was demonstrated that the recipient strains had acquired the ability to produce and be resistant to methylenomycin (12, 21). Transfer of intact linear plasmids containing mercury resistance genes from two Streptomyces strains isolated from the marine environment to S. lividans, conferring mercury resistance to the initially mercury-sensitive recipient, has been reported by Ravel et al. (32). It has also been shown that interspecific transfer of linear plasmids is possible in sterile amended soil microcosms, suggesting that mercury resistance might be spread by plasmid transfer in polluted environments (31).We report here isolation and screening of several thousand actinobacterial strains from the Trondheim fjord (Norway), which resulted in identification of producers of both known and potentially new polyene macrolides with antifungal activity. The ability to produce the polyene macrolide candicidin D was found to be widespread among the Trondheim fjord Streptomyces isolates. We also report that the candicidin biosynthesis genes (can) are present on a linear plasmid identified in one of these isolates, suggesting that the can genes might be spread by means of conjugation.