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.     

Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.     

Structure determination of the UDP-disaccharide fragment of cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum

The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5'-(O-2-acetamido-2-deoxy-beta-manno-pyranuronosyl acid (1----4)-2-acetamido-2-deoxy-alpha-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex.     

Localization of the Miller-Dieker critical region is proximal to locus D17S34 (p144D6) in 17p13.3

A child with normal growth and development and the abnormal karyotype 46,XY,17ps, was analyzed using molecular probes localized to 17p13. The results indicated the presence of two copies of the probes YNZ22.1 (D17S5) and YNH37.3 (D17S28), previously shown to be deleted in all Miller-Dieker (MDS) patients studied. However, the patient was hemizygous for probe p144D6 (D17S34), which is absent in approximately 75% of the MDS patients. As the patient is active at 9 months of age, with no clinical signs of MDS, the results confirm that the absence of locus D17S34 does not lead to the phenotypic expression of MDS. Furthermore, this deletion should assist in defining the distal limits of this contiguous gene syndrome.     

Sequence of the fhuE outer-membrane receptor gene of Escherichia coli K12 and properties of mutants

The fhuE gene of Escherichia coli codes for an outer-membrane receptor protein required for the uptake of iron(III) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77,453, which agrees with the molecular weight of 76,000 determined by polyacrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonB-dependent receptors. A valine to proline exchange in the 'TonB box' abolished transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited increasing iron-coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C-terminal end was exported to the periplasm but failed to be inserted into the outer membrane.     

Aestival dormancy in the cabbage moth Mamestra brassicae L. (Lepidoptera: Noctuidae)

Summary In a geographically wide distribution the life cycles of different populations of the cabbage moth Mamestra brossicae are adapted to a remarkable diversity of climatic conditions. This is undoubtedly a proof of its success in adaptation. Some populations living in regions characterized by a drought period interrupting the growth season are capable of distinguishing between one critical day length signalling the onset of the drought period and another signalling the end of the growth season. This study, therefore, is primarily concerned with the geographical patterns in the variability of the adaptional responses of populations exposed to environmental conditions requiring different strategies and tactics in, synchronizing individual, life cycles. It is also a contribution to our understanding of evolutionary mechanisms maintaining median responses to photoperiodically inductive day lengths in geographically different populations. The populations investigated originated from regions differing in predictability of the incidence, onset and duration of a drought period: Freiburg (48.0°N, Southern Germany), Avignon (44.0°N, Southern France), and Argelès (42.5°N, Southern France). Geographical variation with respect to both onset and duration of a drought period consequently results in clinal variation of the variability of innate day length thresholds triggering aestival dormancy and of innate duration of aestivation. In this paper we considered the influence of geographically changing temperatures on aestival dormancy induction. Even in southern populations of M. brassicae a temperature dependent switch off-mechanism exists which prevents aestival dormancy under certain environmental conditions. The effective temperatures vary geographically, too. What the geographical patterns in adaptive responses really are, is discussed.This research was supported by the Deutsche Forschungsgemeinschaft (Sa 259/3-1)     

A portable EAG system for the measurement of pheromone concentrations in the field

A robust and portable apparatus for the measurement of pheromoneconcentrations under field conditions has been developed. Ituses the insect antenna (Lobesia botrana Hb.) itself as a sensitiveand specific pheromone detector. Shock-proof contact with theelectrodes is maintained by fixing the antenna in a specially-shapedplexiglass holder mounted within a glass tube. This allows measurementsto be made while moving the apparatus. Continuous airflow throughthe tube is generated by a suction pump and the incoming aircan be purified by passage through a charcoal filter. This allowsto readjust the offset and to calibrate the instrument by theapplication of pheromone pulses of known concentrations. Removalof the filter allows the direct access of ambient air over theantenna which responds by generating an electro-antennogram(EAG) as a measure of the pheromone concentration. Using the calibration curve, relative pheromone concentrationsin ambient air in a vineyard can be determined. Sample measurementsfrom areas treated with artificial pheromone for pest controlare presented.