Evolution of green lacewings (Neuroptera: Chrysopidae): a molecular supermatrix approach

We present a time‐calibrated phylogeny of the charismatic green lacewings (Neuroptera: Chrysopidae). Previous phylogenetic studies on the family using DNA sequences have suffered from sparse taxon sampling and/or limited amounts of data. Here we combine all available previously published DNA sequence data and add to it new DNA sequences generated for this study. We analysed these data in a supermatrix using Bayesian and maximum likelihood methods and provide a phylogenetic hypothesis for the family that recovers strong support for the monophyly of all subfamilies and resolves relationships among a large proportion of chrysopine genera. Chrysopinae tribes Leucochrysini and Belonopterygini were recovered as monophyletic sister clades, while the species‐rich tribe Chrysopini was rendered paraphyletic by Ankylopterygini. Relationships among the subfamilies were resolved, although with relatively low statistical support, and the topology varied based on the method of analysis. Greatest support was found for Apochrysinae as sister to Nothochrysinae and Chrysopinae, which is in contrast to traditional concepts that place Nothochrysinae as sister to the rest of the family. Divergence estimates suggest that the stem groups to the various subfamilies diverged during the Triassic‐Jurassic, and that stem groups of the chrysopine tribes diverged during the Cretaceous.     

Isolation of secondary metabolites of Aspergillus ochraceus by HPLC

A method is described for the isolation and purification of ochratoxin A, ochratoxin B, ochratoxin ß mellein, 4-hydroxymellein and penicillic acid produced byAspergillus ochraceus in a synthetic liquid medium. Ochratoxin α, which was not found in the culture medium, was obtained by acid hydrolysis of ochratoxin A. A high pressure liquid Chromatograph equipped with Lichrosorb 100 and Lichrosorb RP-18 columns and UV and/or Refractive Index detectors was used.     

Cytostatic drug testing in human leukemias by means of multiparametric flow cytometry

Human bone marrow cells from 20 patients as well as the permanent human B-cell lines RPMI 1788, Raji, Daudi, T-cell lines Molt, CEM, Jurkat and the promyelocytic line HL 60 were assayed by means of a newly developed in vitro flow cytometric cytostatic drug assay. The cells were exposed to cytosine-arabinoside, L-asparaginase, daunorubicin, prednisone or vincristine. Surviving cells were stained after an incubation period of 2 to 7 days with esterase and pH-indicator dye ADB (1,4-diacetoxy-2,3-dicyanobenzene), dead cells with DNA-dye PI (propidium iodide). Dose-response curves were established using percent surviving cells. It was possible to evaluate bone marrow samples from 16 out of 20 patients. Seven samples were leukemic (acute myeloid leukemia (AML) n = 6, Non-Hodgkin's Lymphoma (NHL) n = 1). Nine samples were from patients either in complete remission or with benign diseases. Daunorubicin and cytosine-arabinoside were cytotoxic in both groups, whereas vincristine was effective mainly in the leukemic group (p less than 0.05). There was significant heterogeneity in the reactivity of AML-marrow cells from different patients to different drugs. The cell lines exhibited different patterns of sensitivity. Vincristine arrested cells in G2/M-phase, cytosine-arabinoside caused an increase of cells in the S-phase.     

Immunocytochemical detection of p21ras expression in fresh human leukaemic cells and cell lines

The expression of p21ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21ras could be detected in most of the cell lines, no significant p21ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state.     

Cleavage of a hydrophilic C-terminal domain increases growth-inhibitory activity of oncostatin M.

Oncostatin M is a polypeptide cytokine, produced by normal and malignant hematopoietic cells, that has several in vitro activities, including the ability to inhibit growth of cultured carcinoma cells. Here we present a structural and functional comparison of two oncostatin M-related proteins (Mr 36,000 and 32,000) secreted by COS cells transfected with oncostatin M cDNA. The smaller of these forms lacked a hydrophilic C-terminal domain comprising predominantly basic amino acids. This domain was also absent from native oncostatin M produced by U937 cells. The 32,000-Mr form of oncostatin M was not produced by cells transfected with plasmids (G195 and G196) in which a potential trypsinlike cleavage site within the hydrophilic C-terminal domain was altered by site-directed mutagenesis. A 32,000-Mr fragment was produced by trypsin treatment of the 36,000-Mr form of oncostatin M. These observations suggest that the 32,000-Mr form of oncostatin M was derived from the 227-amino-acid propeptide by proteolytic cleavage at or near the paired basic residues at positions 195 and 196. Pro-oncostatin M was equally active in radioreceptor assays as the processed form but was 5- to 60-fold less active in growth inhibition assays. Likewise, nonprocessed mutant protein encoded by plasmid G196 was equally active in the radioreceptor assays as the processed form but was five- to ninefold less active in growth inhibition assays. Thus, the highly charged C-terminal domain of pro-oncostatin M is not required for receptor binding or growth-inhibitory activity but may alter the functional properties of the molecule. Propeptide processing of oncostatin M may be important for regulating in vivo activities of this cytokine.     

Mechanism of action of levonorgestrel: in vitro metabolism and specific interactions with steroid receptors in target organs.

Levonorgestrel (LNG) is a synthetic steroid that displays potent progestional and androgenic effects but it lacks estrogen-like activity. To examine the mode of action of this progestin, we studied its metabolism in vitro in target organs and the specific interactions of LNG and its metabolites with putative steroid receptors. The results demonstrated that [³H]LNG was efficiently converted to A-ring reduced derivatives when incubated with rat hypothalamus and pituitary. Under optimal incubation conditions, [³H]5-dihydro LNG (5-LNG) and [³H]3-5-tetrahydro LNG (3,5-LNG) were identified as the major metabolic conversion products, while [³H]3ß, 5-LNG formation occured to a lesser extent. A-ring reduction of LNG was NADPH-dependent. Assessment of the relative binding affinities of LNG and its derivatives to progesterone (PR), androgen (AR) and estrogen (ER) receptors by displacement analysis revealed that unchanged LNG binds with high affinity to PR and AR but not to ER. 5-LNG exhibited a diminished though significant interactions with PR and an enhanced binding affinity for AR as compared with LNG, indicating that 5-reduction of LNG increases its affinity for AR. The most striking finding was that further reduction of the 5-LNG molecule at C-3 abolished its binding activity to PR, AR, and even to ER. The overall data provides a plausible explanation for the lack of estrogen agonistic action of LNG and for its potent progestational and androgenic effects.     

Correlation between ventilation and brain blood flow during hypoxic sleep

Ventilation and brain blood flow (BBF) were simultaneously measured during carbon monoxide (CO) inhalation in awake and sleeping goats up to HbCO levels of 40%. Unilateral BBF, which was continuously measured with an electromagnetic flow probe placed around the internal maxillary artery, progressively increased with CO inhalation in the awake and both sleep stages. The increase in BBF with CO inhalation during rapid-eye-movement (REM) sleep (delta BBF/delta arterial O2 saturation = 1.34 +/- 0.27 ml X min-1 X %-1) was significantly greater than that manifested during wakefulness (0.87 +/- 0.14) or slow-wave sleep (0.92 +/- 0.13). Ventilation was depressed by CO inhalation during both sleep stages but was unchanged from base-line values in awake goats. In contrast to slow-wave (non-REM) sleep, the ventilatory depression of REM sleep was primarily due to a reduction in tidal volume. Since tidal volume is more closely linked to central chemoreceptor function, we believe that these data suggest a possible role of the increased cerebral perfusion during hypoxic REM sleep. Induction of relative tissue alkalosis at the vicinity of the medullary chemoreceptor may contribute to the ventilatory depression exhibited during this sleep period.     

Cyclic AMP levels during induction and repression of cellulase biosynthesis in Thermomonospora curvata.

Specific cellulase production rates (SCPR) were compared with intracellular cyclic AMP (cAMP) levels in the thermophilic actinomycete, Thermomonospora curvata, during growth on several carbon sources in a chemically defined medium. SCPR and cAMP levels were 0.03 U (endoglucanase [EG] units) and 2 pmol per mg of dry cells, respectively, during exponential growth on glucose. These values increased to about 6 and 25, respectively, during growth on cellulose. Detectable EG production ceased when cAMP levels dropped below 10. Cellobiose (usually considered to be a cellulase inducer) caused a sharp decrease in cAMP levels and repressed EG production when added to cellulose-grown cultures. 2-deoxy-D-glucose, although nonmetabolizable in T. curvata, depressed cAMP to levels observed with glucose, but unlike glucose, the 2DG effect persisted until cells were washed and transferred to fresh medium. SCPR values and cAMP levels in cells grown in continuous culture under conditions of cellobiose limitation were markedly influenced by dilution rate (D). The maxima for both occurred at D = 0.085 (culture generation time of 11.8 h). When D was held constant and cellobiose concentration was increased over a 14-fold range to support higher steady state population levels, SCPR values decreased about fivefold, indicating that extracellular catabolite accumulation may be a factor in EG repression. The role of cAMP in the mechanism of this repression appears to be neither simple nor direct, since large changes (up to 200-fold) in SCPR accompany relatively small changes (10-fold) in cellular cAMP levels.