Neural coding in antennal olfactory cells of tsetse flies (Glossina spp.)

Spike trains from individual antennal olfactory cells of tsetse flies (Glossina spp.) obtained during steady-state conditions (spontaneous as well as during stimulation with 1-octen-3-ol) and dynamic stimulation with repetitive pulses of 1-octen-3-ol were investigated by studying the spike frequency and the temporal structure of the trains. In general, stimulation changes the intensity of the spike activity but leaves the underlying stochastic structure unaffected. This structure turns out to be a renewal process. The only independently varying parameter in this process is the mean interspike interval length, suggesting that olfactory cells of tsetse flies may transmit information via a frequency coding. In spike records with high firing rates, however, the stationary records had significant negative first- order serial correlation coefficients and were non-renewal. Some cells in this study were capable of precisely encoding the onset of the odour pulses at frequencies up to at least 3 Hz. Cells with a rapid return to pre-stimulus activity at the end of stimulation responded more adequately to pulsed stimuli than cells with a long increased spike frequency. While short-firing cells process information via a frequency code, long-firing cells responded with two distinctive phases: a phasic, non-renewal response and a tonic, renewal response which may function as a memory of previous stimulations.      

Chloroxine overrides DNA damage tolerance to restore platinum sensitivity in high-grade serous ovarian cancer

High-grade serous cancer (HGSC) accounts for ~67% of all ovarian cancer deaths. Although initially sensitive to platinum chemotherapy, resistance is inevitable and there is an unmet clinical need for novel therapies that can circumvent this event. We performed a drug screen with 1177 FDA-approved drugs and identified the hydroxyquinoline drug, chloroxine. In extensive validation experiments, chloroxine restored sensitivity to both cisplatin and carboplatin, demonstrating broad synergy in our range of experimental models of platinum-resistant HGSC. Synergy was independent of chloroxine’s predicted ionophore activity and did not relate to platinum uptake as measured by atomic absorption spectroscopy. Further mechanistic investigation revealed that chloroxine overrides DNA damage tolerance in platinum-resistant HGSC. Co-treatment with carboplatin and chloroxine (but not either drug alone) caused an increase in γH2AX expression, followed by a reduction in platinum-induced RAD51 foci. Moreover, this unrepaired DNA damage was associated with p53 stabilisation, cell cycle re-entry and triggering of caspase 3/7-mediated cell death. Finally, in our platinum-resistant, intraperitoneal in vivo model, treatment with carboplatin alone resulted in a transient tumour response followed by tumour regrowth. In contrast, treatment with chloroxine and carboplatin combined, was able to maintain tumour volume at baseline for over 4 months. In conclusion, our novel results show that chloroxine facilitates platinum-induced DNA damage to restore platinum sensitivity in HGSC. Since chloroxine is already licensed, this exciting combination therapy could now be rapidly translated for patient benefit.Subject terms: Ovarian cancer, High-throughput screening     

The cockroach heart as a bioassay organ

Detailed procedures are presented for denervation of the American cockroach heart, Periplaneta americana L., by removal of the lateral cardiac nerve cords. Results of bioassay with the innervated heart are also presented and compared to responses obtained from identical assay conditions with the denervated heart.The response of the innervated heart to 10^-3 M sodium azide, slight changes in the concentration of sodium ion, reduced glutathione, saline dilutions, and low amounts of ethanol and acetone was characterized by the immediate appearance of irregularities in the heartbeat. In contrast, the responses of the denervated heart to the first three of these compounds were always characterized by a smooth even heartbeat changing gradually in amplitude and/or rate and/or contractile state of the alary muscles. All assay conditions examined caused qualitatively less response in the beat of the denervated heart.Irregularities in the beat of the innervated heart which were induced in bioassay were found to be due to extreme sensitivity of the spontaneously active cardiac neurons.

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Zusammenfassung Das isolierte abdominale Herz von Periplaneta americana L. reagiert auf Durchspülung mit 10^-3 Mol Natriumazid in physiologischer Kochsalzlösung mit einem unmittelbaren Anstieg des Tonus bis zu fast systolischer Hemmung. Danach verringert das Herz den Tonus allmählich, schlägt unregelmäßig und tritt in eine kurze Periode diastolischer Pause ein, bevor es in Diastole stehen bleibt. Die Herzschlagaktivität wurde wieder aufgenommen, wenn das Natriumazid durch Spülung mit physiologischer Kochsalzlösung ersetzt wird.Nach Entfernung beider Paare der Herzseitennerven gab das Herz von P. americana auf Bespülung mit 10^-3 Mol Natriumazid in physiologischer Kochsalzlösung keine Initialreaktion. Statt dessen stieg der Herzschlag nach 2 min allmählich leicht an und die Herzschlagamplitude nahm im Verlaufe von 6 min sehr allmählich ab. Nach 6 min blieb das Herz in Diastole stehen.Die Herzreaktionen der amerikanischen Küchenschabe waren bei Auftropf-Tests mit anormaler Natriumchloridkonzentration, herabgesetztem Glutathion und Lösungsmitteln ähnlich den für Natriumazid beschriebenen. Die Reaktion des denervierten Herzens ist durch das Fehlen von Unregelmäßigkeiten im Herzschlagmuster gekennzeichnet.Sowohl das denervierte wie das innervierte Herz reagieren auf Testtropfen von nur etwas anormalen Calciumchloridkonzentrationen.

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Supported by USPHS Training grant PHS GM 1076.     

Biochemical characterization of murine leukaemia inhibitory factor produced by Krebs ascites and by yeast cells

A cDNA library was constructed using mRNA from Krebs ascites tumor cells that was shown by Northern blot hybridization to contain mRNA for murine leukemia inhibitory factor (LIF). This library was screened with an oligonucleotide corresponding to the 3' end of a partial LIF cDNA clone, and an overlapping cDNA clone isolated. Nucleotide sequence analysis of this latter clone allowed the complete sequence of LIF to be derived. A cDNA molecule encoding the entire mature LIF protein was installed in a yeast expression vector, and LIF produced up to about 100 ng/ml in the growth medium. The LIF produced by yeast cells has the same biologic properties as native LIF and competes with native 125I-LIF for binding to specific cellular receptors. Two forms of native LIF, distinguishable by their chromatographic behavior on DEAE-Sepharose, were converted by neuraminidase treatment to a form with similar chromatographic behavior, suggesting that the major difference between these two species is the content of sialic acid on the carbohydrate portion. Moreover, yeast-derived recombinant LIF appears to display a different pattern of glycosylation to both forms of native LIF. From in vitro experiments, we conclude that the nature of the glycosylation is not crucial to biologic activity.     

The Wellcome Foundation lecture, 1986. The molecular control of normal and leukaemic granulocytes and macrophages

The development of semisolid culture methods supporting the clonal proliferation and maturation of granulocytes and macrophages led to the discovery of a group of specific glycoproteins, the colony-stimulating factors (CSFs), whose function it is to control the proliferation and functional activity of granulocytes, macrophages and associated blood cells. The four known CSFs in the mouse and man have been purified and complementary DNAs (cDNAs) for each have been cloned. The injection of bacterially synthesized recombinant CSF into mice has demonstrated that these CSFs can function in vivo to regulate granulocyte and macrophage formation. A major physiological role played by these CSFs is to control resistance to invading microorganisms through mechanisms capable of extremely rapid activation. Because the CSFs are the only known proliferative factors for these cells, the CSFs are involved in the initiation and the emergence of myeloid leukaemia but, conversely, at least one of the CSFs, G-CSF, is able to suppress myeloid leukaemic populations because of the ability of the CSFs to initiate differentiation commitment in responding granulocytic and macrophage populations. The CSFs are promising agents for clinical use in the treatment of infections in patients with depressed granulocyte-macrophage formation and possibly in the management of some types of myeloid leukaemia.     

Genomic and phenotypic differentiation among Methanosarcina mazei populations from Columbia River sediment

Methanogenic archaea are genotypically and phenotypically diverse organisms that are integral to carbon cycling in anaerobic environments. Owing to their genetic tractability and ability to be readily cultivated, Methanosarcina spp. have become a powerful model system for understanding methanogen biology at the cellular systems level. However, relatively little is known of how genotypic and phenotypic variation is partitioned in Methanosarcina populations inhabiting natural environments and the possible ecological and evolutionary implications of such variation. Here, we have identified how genomic and phenotypic diversity is partitioned within and between Methanosarcina mazei populations obtained from two different sediment environments in the Columbia River Estuary (Oregon, USA). Population genomic analysis of 56 M. mazei isolates averaging <1% nucleotide divergence revealed two distinct clades, which we refer to as ‘mazei-T'' and ‘mazei-WC''. Genomic analyses showed that these clades differed in gene content and fixation of allelic variants, which point to potential differences in primary metabolism and also interactions with foreign genetic elements. This hypothesis of niche partitioning was supported by laboratory growth experiments that revealed significant differences in trimethylamine utilization. These findings improve our understanding of the ecologically relevant scales of genomic variation in natural systems and demonstrate interactions between genetic and ecological diversity in these easily cultivable and genetically tractable model methanogens.     

Studies on colony formation in vitro by mouse bone marrow cells. II. Action of colony stimulating factor

An analysis was made of some of the processes involved in the stimulation by colony stimulating factor (CSF) of cluster and colony formation by mouse bone marrow cells in agar cultures in vitro. Colony formation was shown to be related to the concentration and not the total amount of CSF. The concentration of CSF determined the rate of new cluster initiation in cultures and the rate of growth of individual clusters. Colony growth depleted the medium of CSF suggesting that colony cells may utilise CSF during proliferation. Bone marrow cells incubated in agar in the absence of CSF rapidly died or lost their capacity to proliferate and form clusters or colonies. CSF appears (a) to be necessary for survival of cluster-and colony-forming cells or for survival of their proliferative potential, (b) to shorten the lag period before individual cells commence proliferation and (c) to increase the growth rate of individual clusters and colonies.