The sigma-1 receptor is enriched in postsynaptic sites of C-terminals in mouse motoneurons. An anatomical and behavioral study.

The sigma-1 receptor regulates various ion channel activity and possesses protein chaperone function. Using an antibody against the full sequence of the sigma-1 receptor we detected immunostaining in wild type but not in knockout mice. The receptor was found primarily in motoneurons localized to the brainstem and spinal cord. At the subcellular level the receptor is restricted to large cholinergic postsynaptic densities on the soma of motoneurons and is colocalized with the Kv2.1 potassium channel and the muscarinic type 2 cholinergic receptor. Ultrastructural analysis of the neurons indicates that the immunostained receptor is located close but separate from the plasma membrane, possibly in subsurface cisternae formed from the endoplasmic reticulum (ER), which are a prominent feature of cholinergic postsynaptic densities. Behavioral testing on a rotorod revealed that Sigma-1 receptor knockout mice remained on the rotorod for significantly less time (a shorter latency period) compared to the wild type mice. Together these data indicate that the sigma-1 receptor may play a role in the regulation of motor behavior.

Inducible resistance to oxidant stress in the protozoan Leishmania chagasi.

Leishmania sp. protozoa are introduced into a mammalian skin by a sandfly vector, whereupon they encounter increased temperature and toxic oxidants generated during phagocytosis. We studied the effects of 37 degrees C "heat shock" or sublethal menadione, which generates superoxide and hydrogen peroxide, on Leishmania chagasi virulence. Both heat and menadione caused parasites to become more resistant to H(2)O(2)-mediated toxicity. Peroxide resistance was also induced as promastigotes developed in culture from logarithmic to their virulent stationary phase form. Peroxide resistance was not associated with an increase in reduced thiols (trypanothione and glutathione) or increased activity of ornithine decarboxylase, which is rate-limiting in trypanothione synthesis. Membrane lipophosphoglycan increased in size as parasites developed to stationary phase but not after environmental exposures. Instead, parasites underwent a heat shock response upon exposure to heat or sublethal menadione, detected by increased levels of HSP70. Transfection of promastigotes with L. chagasi HSP70 caused a heat-inducible increase in resistance to peroxide, implying it is involved in antioxidant defense. We conclude that leishmania have redundant mechanisms for resisting toxic oxidants. Some are induced during developmental change and others are induced in response to environmental stress.

Short report: regulation of inducible heat shock protein 70 genes in Leishmania chagasi.

Eukaryotic and prokaryotic cells undergo an increase in heat shock proteins, including hsp70, during exposure to environmental stress and during some developmental changes. In trypanosomatid protozoa such as Leishmania sp. that cycle between poikilothermic vectors and mammalian hosts, this heat shock response occurs at programmed times in the parasite's life cycle. The increase in heat shock proteins in mammalian cells is initiated by an increased rate of transcription, resulting in greater amounts of total hsp70 RNA and protein. In contrast, we found a dramatic increase in hsp70 RNA during growth of Leishmania chagasi promastigotes from logarithmic to stationary phase in liquid culture, which was not accompanied by an increased amount of hsp70 protein. Furthermore, there was a 1.8-fold increase in hsp70 protein induced by exposure of L. chagasi to superoxide, but this was not associated with an increase in hsp70 RNA. We conclude that in contrast to higher eukaryotes, the amount of hsp70 protein produced by Leishmania sp. is not regulated by the steady state level of total hsp70 RNA.

N-tert-butyl-alpha-phenylnitrone: a free radical trap with unanticipated effects on diaphragm function.

The spin trap N-tert-butyl-alpha-phenylnitrone (PBN) has a high avidity for free radical species and hence functions as an antioxidant in many biological systems. As such, we hypothesized that PBN would have powerful antioxidant effects on muscle function. We examined the effects of PBN on directly stimulated in vitro (37 degrees C) rat diaphragm. First, a dose-response curve for the effects of PBN on force frequency (n = 8) was established by comparing PBN-treated muscle strips (0.01-10 mM) with time- and stimulus-matched control strips. Second, the effect of 1.0 mM PBN on muscle endurance (n = 8) was established. Our findings were as follows. 1) Compared with baseline, peak twitch and low-frequency muscle tensions increased in a dose-dependent fashion, with peak effects at 1.0 mM PBN. 2) Muscle function at all stimulation frequencies was depressed at doses above 1.0 mM PBN. 3) Complete inhibition at 10 mM PBN was reversed with caffeine administration or washout. 4) During early fatigue, 1.0 mM PBN facilitated force. However, endurance time decreased in the PBN-treated group. We conclude that PBN has direct reversible dose-dependent effects on diaphragm function. However, facilitation of low-frequency forces and the lack of fatigue-attenuating properties suggest that PBN has atypical antioxidant effects on muscle function.

Response of Leishmania chagasi promastigotes to oxidant stress.

At the onset of infection, Leishmania promastigotes are phagocytized by mammalian macrophages. They must survive despite exposure to toxic oxidants such as hydrogen peroxide (H2O2) and superoxide (.O2-) generated during phagocytosis. We investigated the effects of these oxidants on Leishmania chagasi promastigotes and promastigote mechanisms for oxidant resistance. According to spin trapping and electron paramagnetic resonance spectrometry, .O2- could be generated by exposure of promastigotes to the redox-cycling compound menadione. Incubation in either menadione or H2O2 caused a concentration-dependent loss of promastigote viability. However, incubation in sublethal concentrations of H2O2 or menadione caused a stress response in promastigotes. This oxidant-induced response was associated with an increase in the amount of heat shock protein hsp70. Induction of a stress response by exposure of promastigotes either to heat shock or to sublethal oxidants (H2O2 or menadione) caused promastigotes to become more resistant to H2O2 toxicity. Sublethal menadione also caused promastigotes to become more virulent in a BALB/c mouse model of leishmaniasis. We previously correlated H2O2 cytotoxicity for promastigotes with the formation of hydroxyl radical (.OH) from H2O2. However, according to electron paramagnetic resonance spectrometry, the increase in H2O2 resistance after exposure to sublethal oxidants was not associated with diminished generation (i.e., scavenging) of .OH. These data suggest that there is a cross-protective stress response that occurs after exposure of L. chagasi promastigotes to heat shock or to sublethal H2O2 or .O2-, exposures that also occur during natural infection. This response results in increased resistance to H2O2 toxicity and increased virulence for a mammalian host.

Acquisition of iron from transferrin and lactoferrin by the protozoan Leishmania chagasi.

Leishmania chagasi, the cause of South American visceral leishmaniasis, requires iron for its growth. However, the extent to which different iron sources can be utilized by the parasite is not known. To address this question, we studied acquisition of iron from lactoferrin and transferrin by the extracellular promastigote form of L. chagasi during growth in vitro. A promastigote growth medium based on minimal essential medium supplemented with iron-depleted serum supported promastigote growth only after the addition of exogenous iron. The addition of 8 microM iron chelated to lactoferrin or hemin resulted in normal promastigote growth. Ferritransferrin also supported promastigote growth, but only after a considerable lag. Promastigotes grown in all three iron sources generated similar amounts of hydroxyl radical upon exposure to hydrogen peroxide, indicating that none of these protected parasites against generation of this toxic radical. Promastigotes were able to take up 59Fe chelated to either transferrin or lactoferrin, although uptake from 59Fe-lactoferrin occurred more rapidly. 59Fe uptake from either 59Fe-transferrin or 59Fe-lactoferrin was inhibited by a 10-fold excess of unlabeled ferrilactoferrin, ferritransferrin, apolactoferrin, apotransferrin, or iron nitrilotriacetate but not ferritin or bovine serum albumin. There was no evidence for a role for parasite-derived siderophores or proteolytic cleavage of ferritransferrin or ferrilactoferrin in the acquisition of iron by promastigotes. Thus, L. chagasi promastigotes can acquire iron from hemin, ferrilactoferrin, or ferritransferrin. This capacity to utilize several iron sources may contribute to the organism's ability to survive in the diverse environments it encounters in the insect and mammalian hosts.

[Distance diagnosis of skin diseases]. / Fjerndiagnostisering av hudsykdommer.

During a period of eight weeks in late 1989, doctors in general practice in Kirkenes brought 27 dermatological patients to a studio at the local hospital. Video images were transmitted via a two-way telephone and video network, which enabled the patient and the doctor in Kirkenes to consult a specialist in dermatology in Tromsø. All the patients received written information before the consultation. Anamnestic information and clinical manifestations were presented from the studio at Kirkenes sykehus. The dermatologist diagnosed the disease and prescribed treatment from the studio at the hospital in Tromsø. The technical equipment satisfied our demands for remote diagnostics in dermatology. All the patients were positive to remote consultations.