The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education.

Natural killer (NK) cell responsiveness in the mouse is determined in an education process guided by inhibitory Ly49 and NKG2A receptors binding to MHC class I molecules. It has been proposed that inhibitory signalling in human NK cells involves Abl-1 (c-Abl)-mediated phosphorylation of Crk, lowering NK cell function via disruption of a signalling complex including C3G and c-Cbl, suggesting that NK cell education might involve c-Abl. Mice deficient in c-Abl expression specifically in murine NK cells displayed normal inhibitory and activating receptor repertoires. Furthermore, c-Abl-deficient NK cells fluxed Ca2+ normally after triggering of ITAM receptors, killed YAC-1 tumour cells efficiently and showed normal, or even slightly elevated, capacity to produce IFN-γ after activating receptor stimulation. Consistent with these results, c-Abl deficiency in NK cells did not affect NK cell inhibition via the receptors Ly49G2, Ly49A and NKG2A. We conclude that signalling downstream of murine inhibitory receptors does not involve c-Abl and that c-Abl plays no major role in NK cell education in the mouse.

Actin filament polymerization regulates gliding motility by apicomplexan parasites.

Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric. In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites. To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin. JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane. Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility. However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry. Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells. These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa.

Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170.

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170's putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

Glucocorticoid receptor mRNA in Alzheimer's diseased hippocampus.

Hypothalamic-pituitary-adrenal (HPA) axis dysfunction is a common finding in Alzheimer's dementia. Since there is a loss of hippocampal corticosteroid receptors in animal models of aging, and since hippocampal cell loss occurs in Alzheimer's disease (AD), it has been suggested that a loss of hippocampal glucocorticoid receptors (GR) may underlie some aspects of HPA axis dysfunction in patients with AD. Levels of corticosteroid receptor protein are not reliably determined in postmortem human brain due to rapid lability. In contrast, levels of mRNA coding for GR are stable in postmortem tissue. We report here initial observations from in situ hybridization experiments which indicate that regional levels of glucocorticoid receptor mRNA in hippocampus, as determined by film autoradiography, are significantly higher in AD hippocampus than in controls. While neuronal levels of GR mRNA in AD, revealed by emulsion autoradiography, were equal in control and AD tissue. Taken together these results suggest that adrenal dysfunction in AD may relate to defects in receptor function rather than corticosteroid receptor loss in the hippocampus.

Postmortem stability of mRNA for glucocorticoid and mineralocorticoid receptor in rodent brain.

The relative postmortem stability of the mRNA's for glucocorticoid (GR) and mineralocorticoid (MR) receptor in rodent brain was determined using semi-quantitative in situ hybridization (ISH). Rats were killed by CO2 asphyxiation and their brains removed immediately (0 h) or following 12 h or 24 h delays. Specific hybridization of GR and MR anti-sense [35S]RNA-probe to tissue mRNA encoding these receptors was detected using film and emulsion autoradiography. The most intense labeling for GR mRNA was in the dentate gyrus followed by the CA1 hippocampal region. Lower, but still detectable signal, was apparent over CA3-CA4 pyramidal cell regions. MR mRNA was detected throughout the CA1-4 pyramidal cell fields of the hippocampus and the granular cells of the dentate gyrus. Film images demonstrated that even in the 24 h postmortem delay group intense specific signal was present in sections hybridized with both anti-sense GR and MR probes, although there was some diminution in signal intensity in cortical areas at this later postmortem delay. These initial experiments with rat brain demonstrate that the mRNA's for both GR and MR, as detected with ISH, are stable for up to 24 h following death.

Fibrillation and accelerated AChR degradation in long-term muscle organ culture.

Evaluation of the precise molecular dynamics of endplate maintenance and reorganization has been limited by the lack of available in vitro preparations. We describe an organ culture preparation of mouse diaphragm muscle which permits long-term maintenance of muscle viability. Spontaneous fibrillations, increased levels of extrajunctional acetylcholine receptors, accelerated rates of junctional acetylcholine receptor turnover and maintenance of fine structure of denervated mouse diaphragm muscle in organ culture was evaluated under different culture conditions. Of several standard tissue culture media tested with and without fetal calf serum, medium 199 plus fetal calf serum was best for maintaining this muscle for greater than 2 weeks. The serum component could be partially eliminated by addition of non-glucose energy substrates such as D-beta-hydroxybutyric acid and L-glutamine. This preparation will permit a more controlled examination of the molecular components of endplate diseases.

Long-term cultures of neurons from adult frog brain express GABA and glutamate-activated channels.

Cells from adult Xenopus laevis brainstem and spinal cord were dissociated with mild enzymatic treatment and grown in long-term cell culture. These cells had specific attachment/substrate and medium/serum requirements. Cells with bipolar and multipolar morphology were positively identified as neurons using immunohistochemistry with monoclonal antibodies to rat and bovine neurofilament proteins which we show here cross-react with similar amphibian proteins. Patch clamp recordings demonstrated that these neurons have populations of ionic channels which are activated by L-glutamate or gamma-aminobutyric acid (GABA). The characteristics of these channels were similar to those previously described for GABA- and glutamate-activated channels in embryonic mammalian neurons isolated in culture. Cell cultures of neurons isolated from adult Xenopus laevis brain may be a useful and simple preparation with which to examine the modulation of neuronal properties by various agents over longer time intervals then has been previously possible.

5 alpha-dihydrotestosterone has nonspecific effects on membrane channels and possible genomic effects on ACh-activated channels.

1. Myotubes cultured from adult male Xenopus laevis laryngeal muscle have been found to express androgen receptors. 2. Using patch-clamp techniques, it was found that 5 alpha-dihydrotestosterone (5 alpha-DHT) can act directly at membranes of these myotubes to alter the kinetic properties of acetylcholine- (ACh) activated single channels. 3. When the culture medium contains 5 alpha-DHT for greater than 6 days, the androgen acts through its receptors to markedly increase ACh-activated, single-channel conductances and alter single-channel kinetics. These effects do not occur if the antiandrogen, flutamide, which prevents the androgen from combining with its receptor, is added to the medium before the addition of the 5 alpha-DHT. These effects also do not occur in myotubes cultured from quadriceps femoris muscles that have much lower levels of specific androgen receptor. 4. When alpha-bungarotoxin (alpha-BTX) is added to the medium for 3 h before recording, no ACh channels can be recorded from myotubes in control medium within 5 h after washout of the alpha-BTX. However, when the culture medium contains 5 alpha-DHT for greater than or equal to 6 days, ACh channels can be recorded within 8 min of the washout of the alpha-BTX. 5. The results suggest that 5 alpha-DHT may act to alter the properties of ACh-activated ion channels at multiple sites in excitable cells.

A proposed neural pathway for vocalization in South African clawed frogs, Xenopus laevis.

Vocalizations of South African clawed frogs are produced by contractions of laryngeal muscles innervated by motor neurons of the caudal medulla (within cranial nerve nucleus IX-X). We have traced afferents to laryngeal motor neurons in male and female frogs using retrograde axonal transport of horseradish peroxidase conjugated to wheat germ agglutinin (HRP-WGA). After iontophoretic injection of HRP-WGA into n. IX-X, retrogradely labelled neurons were seen in the contralateral n. IX-X, in rhombencephalic reticular nuclei, and in the pre-trigeminal nucleus of the dorsal tegmental area (DTAM) of both males and females.

Androgen and gonadotropin effects on male mate calls in South African clawed frogs, Xenopus laevis.

Mate calling is a prominent reproductive behavior of male South African clawed frogs. Calls consist of alternating slow- and fast-amplitude-modulated trills. Each trill is made up of a series of clicks. The effects of administration of exogenous gonadotropin and androgen on mate calling were studied in male Xenopus laevis. Males were paired with unreceptive female frogs to elicit maximal calling. The amount of time each animal spent calling during the testing period, the peak fundamental frequency of the calls, the rate of calling, and the interclick interval (ICI, a measure of the temporal patterning of the calls) were measured in intact, castrated, and hormone-replaced frogs. Injection of human chorionic gonadotropin (HCG) into intact frogs increased the amount of time spent calling and the ICI relative to measures taken after water injection. Castrated males did not call even when given HCG. Testosterone and dihydrotestosterone treatment reinstated calling in castrates and increased circulating levels of androgens. When androgen-replaced castrated males were injected with HCG, the amount of time spent calling increased and approached levels of intact, HCG-injected males. The above results suggest that androgens are necessary for the production of calls. Gonadotropins appear to play an important role in mate calling, a role at least partly independent of effects on testicular androgen synthesis.

Central control of ultrasonic vocalizations in neonatal rats: I. Brain stem motor nuclei.

Neonatal rats produce ultrasonic vocalizations in response to cold stress. The rate and intensity of these vocalizations decrease dramatically as the pup reaches maturity. The laryngeal nerves controlling the production of ultrasounds and their nuclei of origin were investigated in 10-day old rat pups. Unilateral and bilateral transections of the inferior laryngeal nerve reduced ultrasounds to undetectable levels. Transecting the superior laryngeal nerve either unilaterally or bilaterally reduced the sound pressure level, reduced the rate, and increased the fundamental frequency of the ultrasounds. Retrograde transport of horseradish peroxidase revealed that the efferent axons in the inferior laryngeal nerve arise from the cells of the dorsal formation of the nucleus ambiguus and that efferents in the superior laryngeal nerve originate from the ventral formation of the nucleus ambiguus. Therefore, different aspects of the production of pup ultrasounds appear to be controlled by distinct neuronal subpopulations of the nucleus ambiguus.