Highly specialized Cretaceous beetle parasitoids (Ripiphoridae) identified with optimized visualization of microstructures

Extremely miniaturized longipedes insects (body length c. 0.3 mm) embedded in two pieces of Cretaceous amber from Myanmar are described and interpreted. Using inverted fluorescence and light microscopy for detailed analysis of microstructures, the inclusions were identified as primary larvae of the beetle family Ripiphoridae, subfamily Ripidiinae. While the structure of thoracic and abdominal segments including appendages corresponds well with the groundplan known in recent members of Ripidiinae, a curved prosternal ridge with prominent spines (each c. 5 μm), the reduced condition of stemmata and antennae and the lack of sharp mandibles are unique features within the entire family, apparently apomorphies of the longipedes larvae. A sinuate prosternal edge with a dense row of spines (prosternoctenidium) might be homologous with ‘head ctenidia’ in some previously described miniaturized conicocephalate larvae, but further investigation is needed. The morphological differences between the head of longipedes larvae and extant Ripidiinae are interpreted as adaptations to different groups of hosts and life strategies. Palaeoethology of the longipedes larvae is briefly discussed. In addition, the systematic placement of conicocephalate larvae from Canadian, Myanmar and Russian Cretaceous ambers, already interpreted by various authors as primary instars within Coleopterida (assigned to either Strepsiptera or to the coleopteran Tenebrionoidea: Ripiphoridae), is discussed.     

Comparative mineral and hormonal analyses of wild type and TLS somaclonal variant derived from oil palm (Elaeis guineensis Jacq. var. tenera) tissue culture

Comparative mineral and hormonal analyses were made on tissue culture derived truncated leaf syndrome and wild type oil palm seedlings. Mineral analysis confirmed that Boron, Zinc and chlorophyll levels were significantly lower in truncated leaf syndrome leaves than those of wild type. Hormonal analysis also revealed various cytokinin derivatives such as trans-zeatin, trans-zeatin riboside, trans-zeatin O-glucoside and trans-zeatin riboside 5??mono phosphate were significantly higher in truncated leaf syndrome leaves compared to wild type leaves. Brassinolide level was also significantly higher in truncated leaf syndrome leaves than those of the wild type. These observations suggest that the truncated leaf syndrome abnormality could be associated to high cytokinin and brassinosteroid production which affects the uptake of Boron and Zinc.     

Engineering and mechanistic studies of the Arabidopsis FAE1 beta-ketoacyl-CoA synthase, FAE1 KCS.

The Arabidopsis FAE1 beta-ketoacyl-CoA synthase (FAE1 KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoAs. Sequence analysis of FAE1 KCS predicted that this condensing enzyme is anchored to a membrane by two adjacent N-terminal membrane-spanning domains. In order to characterize the FAE1 KCS and analyze its mechanism, FAE1 KCS and its mutants were engineered with a His6-tag at their N-terminus, and expressed in Saccharomyces cerevisiae. The membrane-bound enzyme was then solubilized and purified to near homogeneity on a metal affinity column. Wild-type recombinant FAE1 KCS was active with several acyl-CoA substrates, with highest activity towards saturated and monounsaturated C16 and C18. In the absence of an acyl-CoA substrate, FAE1 KCS was unable to carry out decarboxylation of [3-(14)C]malonyl-CoA, indicating that it requires binding of the acyl-CoA for decarboxylation activity. Site-directed mutagenesis was carried out on the FAE1 KCS to assess if this condensing enzyme was mechanistically related to the well characterized soluble condensing enzymes of fatty acid and flavonoid syntheses. A C223A mutant enzyme lacking the acylation site was unable to carry out decarboxylation of malonyl-CoA even when 18:1-CoA was present. Mutational analyses of the conserved Asn424 and His391 residues indicated the importance of these residues for FAE1-KCS activity. The results presented here provide the initial analysis of the reaction mechanism for a membrane-bound condensing enzyme from any source and provide evidence for a mechanism similar to the soluble condensing enzymes.     

3-Ketoacyl-acyl carrier protein synthase III from spinach (Spinacia oleracea) is not similar to other condensing enzymes of fatty acid synthase.

A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated. Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA. A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated. Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III. Northern analysis confirmed that it was expressed in both leaf and root. The clone contained a 1218-bp open reading frame coding for 405 amino acids. The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein. The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E. coli, respectively. It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals.     

Empirical testing of cryoconite granulation: Role of cyanobacteria in the formation of key biogenic structure darkening glaciers in polar regions

Cryoconite, the dark sediment on the surface of glaciers, often aggregates into oval or irregular granules serving as biogeochemical factories. They reduce a glacier's albedo, act as biodiversity hotspots by supporting aerobic and anaerobic microbial communities, constitute one of the organic matter (OM) sources on glaciers, and are a feeder for micrometazoans. Although cryoconite granules have multiple roles on glaciers, their formation is poorly understood. Cyanobacteria are ubiquitous and abundant engineers of cryoconite hole ecosystems. This study tested whether cyanobacteria may be responsible for cryoconite granulation as a sole biotic element. Incubation of Greenlandic, Svalbard, and Scandinavian cyanobacteria in different nutrient availabilities and substrata for growth (distilled water alone and water with quartz powder, furnaced cryoconite without OM, or powdered rocks from glacial catchment) revealed that cyanobacteria bind mineral particles into granules. The structures formed in the experiment resembled those commonly observed in natural cryoconite holes: they contained numerous cyanobacterial filaments protruding from aggregated mineral particles. Moreover, all examined strains were confirmed to produce extracellular polymeric substances (EPS), which suggests that cryoconite granulation is most likely due to EPS secretion by gliding cyanobacteria. In the presence of water as the only substrate for growth, cyanobacteria formed mostly carpet-like mats. Our data empirically prove that EPS-producing oscillatorialean cyanobacteria isolated from the diverse community of cryoconite microorganisms can form granules from mineral substrate and that the presence of the mineral substrate increases the probability of the formation of these important and complex biogeochemical microstructures on glaciers.     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.     

Modulation of adenylate cyclase activity of fibroblasts by free fatty acids and phospholipids

The effect of certain lipids on adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from fibroblasts in culture has been investigated. The unsaturated fatty acids, as well as lysolecithin, were found to act as potent inhibitors of fibroblast adenylate cyclase activity. Increasing the degree of unsaturation increases the extent of inhibition noted at a given fatty acid concentration. The inhibitory effect of the unsaturated fatty acids or lysolecithin is not selective for a specific function of the adenylate cyclase system since basal, and hormone- or fluoride-stimulated cyclase activities are inhibited to the same extent. The fatty acid-inactivated state of fibroblast adenylate cyclase is not readily reversed for enzyme activity is not restored when arachidonate-treated membranes are washed with Tris buffer containing 10 mm EDTA, 0.15 mm albumin, or 0.15 m KCl. Previous studies have shown that the adenylate cyclase system from Moloney sarcoma virus-transformed NRK (MNRK) cells is not stimulated by the addition of GTP or hormones. Of interest is the present finding that the addition of unsaturated fatty acids, or lysolecithin, over a narrow concentration range (0.1 – 0.2 mm) leads to partial restoration of GTP activation of MNRK cyclase activity. Hormonal responsiveness to l-epinephrine or prostaglandin E₁ is not restored to the MNRK enzyme with fatty acid or lysolecithin treatment.     

Topoisomerase mutants and physiological conditions control supercoiling and Z-DNA formation in vivo.

The influence of topoisomerase I and gyrase mutations in Escherichia coli on the supercoiled density of recombinant plasmids and the stability of left-handed Z-DNA was investigated. The formation of Z-DNA in vivo by dC-dG sequences of different lengths was used to determine the effective plasmid supercoil densities in the mutant strains. The presence of Z-DNA in the cells was detected by linking number and EcoRI methylase inhibition assays. A change in the unrestrained superhelical tension in vivo directly effects the B- to Z-DNA transition. Alterations in the internal or external environment of the cells, such as the inactivation of gyrase or topoisomerase I, a gyrase temperature-sensitive mutant, or starvation of cells, have a dramatic influence on the topology of plasmids. Also, E. coli has significantly more superhelical strain than Klebsiella, Morganella, or Enterobacter. These studies indicate that linking deficiency and effective supercoil density are mutually independent variables of plasmid tertiary structure. A variety of factors, such as protein-DNA interactions, activity of topoisomerases, and the resulting supercoil density, contribute to the B to Z transition inside living cells.