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Dogs with a 4-bp deletion in the MDR1(or ABCB1) gene show intolerance to certain drugs routinely used in veterinary medicine, such as ivermectin, vincristine, and doxorubicin. The mutation leads to a dysfunctional P-glycoprotein drug transporter, which results in drug accumulation in the brain and severe neurotoxicity. A rapid and accurate in-house test to determine the genotype of patients in cases of acute neurotoxic signs or in tumor patients is desirable. We describe a cost-effective detection method with simple technical equipment for veterinary practice. Two allele-specific methods are presented, which allow discrimination of all genotypes, require little hands-on time, and show the results within ~1 h after DNA sampling. DNA from buccal swabs of 115 dogs with known genotype (no mutation, n= 54; heterozygous for the mutation, n= 37; homozygous for the mutation, n= 24) was extracted either by using a column-based extraction kit or by heating swabs in a simple NaOH-Tris buffer. Amplification was performed either by allele-specific fast polymerase chain reaction or by allele-specific loop-mediated isothermal amplification (LAMP). Analysis was done either on agarose gels, by simple endpoint visualization using ultraviolet light, or by measuring the increase of fluorescence and time to threshold crossing. Commercial master mixes reduced the preparation time and minimized sources of error in both methods. Both methods allowed the discrimination of all 3 genotypes, and the results of the new methods matched the results of the previous genotyping. The presented methods could be used for fast individual MDR1/ABCB1genotyping with less equipment than existing methods.

The transcription factor SOX2 (3q26.3–q27) is a key regulator of foregut development and an embryonic stem cell factor cooperating during induction of pluripotency in terminally differentiated somatic cells. Recently, we found SOX2 to be amplified in a subset of squamous cell lung and esophageal cancers. The aim of this study was to explore the prognostic role of SOX2 in a large series of squamous cell carcinomas and adenocarcinomas of the lung. A total of 891 samples from two independent population-based cohorts were assessed by fluorescence in situ hybridization and immunohistochemistry. Furthermore, we assessed for associations between SOX2 amplification/upregulation and clinicopathological features. Similar results were found in the two cohorts. Within squamous cell carcinoma cases, 8% high-level as well as 68 and 65% low-level SOX2 amplifications occurred in the two cohorts, respectively. In adenocarcinomas, no high-level amplification was found and low-level amplification occurred in 6% of the two cohorts. Within squamous cell carcinomas of one cohort, SOX2 amplification was associated with lower tumor grade, while higher levels of SOX2 expression were related to younger age, smaller tumor size, and lower probability of angiolymphatic invasion and metastasis. High SOX2 expression levels proved to be a marker for prolonged overall survival among patients with squamous cell carcinomas. In conclusion, SOX2 amplification and upregulation are frequent events in squamous cell carcinomas of the lung and are associated with indicators of favorable prognosis.

The transcription factor SOX2(3q26.3–q27) is a key regulator of foregut development and an embryonic stem cell factor cooperating during induction of pluripotency in terminally differentiated somatic cells. Recently, we found SOX2to be amplified in a subset of squamous cell lung and esophageal cancers. The aim of this study was to explore the prognostic role of SOX2in a large series of squamous cell carcinomas and adenocarcinomas of the lung. A total of 891 samples from two independent population-based cohorts were assessed by fluorescence in situhybridization and immunohistochemistry. Furthermore, we assessed for associations between SOX2amplification/upregulation and clinicopathological features. Similar results were found in the two cohorts. Within squamous cell carcinoma cases, 8% high-level as well as 68 and 65% low-level SOX2amplifications occurred in the two cohorts, respectively. In adenocarcinomas, no high-level amplification was found and low-level amplification occurred in 6% of the two cohorts. Within squamous cell carcinomas of one cohort, SOX2amplification was associated with lower tumor grade, while higher levels of SOX2 expression were related to younger age, smaller tumor size, and lower probability of angiolymphatic invasion and metastasis. High SOX2 expression levels proved to be a marker for prolonged overall survival among patients with squamous cell carcinomas. In conclusion, SOX2amplification and upregulation are frequent events in squamous cell carcinomas of the lung and are associated with indicators of favorable prognosis.

Identification of specific somatic gene alterations is crucial for the insight into the development, progression, and clinical behavior of individual cancer types. The recently discovered recurrent ERG rearrangement in prostate cancer might represent a prostate cancer-specific alteration that has not been systematically assessed in tumors other than prostate cancer. Aim of this study was to assess, whether the ERG rearrangement and the distinct deletion site between TMPRSS2 and ERG, both predominantly resulting in a TMPRSS2–ERG fusion, occur in tumors other than prostate cancer. We assessed 54 different tumor types (2942 samples in total) for their ERG rearrangement status by fluorescence in situ hybridization (FISH). To calibrate, we analyzed 285 prostate cancer samples for the ERG rearrangement frequency. Additionally, we interrogated a high-resolution single nucleotide polymorphism (SNP) data set across 3131 cancer specimens (26 tumor types) for copy number alterations. None of the 54 different tumor types assessed by FISH harbored an ERG rearrangement, whereas the prostate cancer samples revealed an ERG rearrangement in 49.5% of cases. Furthermore, within the 26 tumor types assessed for copy number alterations by SNP, the distinct deletion site between TMPRSS2 and ERG (21q22.2–3) was detectable exclusively in prostate cancer. Although Ewing's sarcoma and AML have known rearrangements rarely involving ERG, we hypothesize that the ERG rearrangement as well as the distinct deletion site on 21q22.2–3 between TMPRSS2 and ERG are prostate-cancer-specific genomic alterations. These observations provide further insight into the oncogenesis of prostate cancer and might be critical for the development of ERG rearrangement assessment as a clinical tool.

Identification of specific somatic gene alterations is crucial for the insight into the development, progression, and clinical behavior of individual cancer types. The recently discovered recurrent ERGrearrangement in prostate cancer might represent a prostate cancer-specific alteration that has not been systematically assessed in tumors other than prostate cancer. Aim of this study was to assess, whether the ERGrearrangement and the distinct deletion site between TMPRSS2and ERG, both predominantly resulting in a TMPRSS2–ERGfusion, occur in tumors other than prostate cancer. We assessed 54 different tumor types (2942 samples in total) for their ERGrearrangement status by fluorescence in situhybridization (FISH). To calibrate, we analyzed 285 prostate cancer samples for the ERGrearrangement frequency. Additionally, we interrogated a high-resolution single nucleotide polymorphism (SNP) data set across 3131 cancer specimens (26 tumor types) for copy number alterations. None of the 54 different tumor types assessed by FISH harbored an ERGrearrangement, whereas the prostate cancer samples revealed an ERGrearrangement in 49.5% of cases. Furthermore, within the 26 tumor types assessed for copy number alterations by SNP, the distinct deletion site between TMPRSS2and ERG(21q22.2–3) was detectable exclusively in prostate cancer. Although Ewing's sarcoma and AML have known rearrangements rarely involving ERG, we hypothesize that the ERGrearrangement as well as the distinct deletion site on 21q22.2–3 between TMPRSS2and ERGare prostate-cancer-specific genomic alterations. These observations provide further insight into the oncogenesis of prostate cancer and might be critical for the development of ERGrearrangement assessment as a clinical tool.

Group II metabotropic glutamate receptors (mGluR2/3) are suggested to modulate anxiety, arousal, and stress including autonomic control. However, no study has investigated mGluR2/3-related effects on baseline autonomic activity and reactivity to emotional challenge in humans as yet. Using a double-blind, randomized placebo-controlled, cross-over study design, we investigated the influence of a 1-week treatment with the mGluR2/3agonist LY544344, prodrug of LY354740, on autonomic reactivity to a cholecystokinin tetrapeptide (CCK-4) panic challenge in eight healthy young men. The main outcome measures were time and frequency domain heart rate variability parameters during baseline, CCK-4 challenge, and recovery. There was no evidence for LY544344-mediated effects on baseline and CCK-4 challenge vagal activity, but a significantly lower recovery low frequency (%) and low frequency/high frequency ratio in the LY544344 group, suggesting enhanced autonomic recovery. This pilot study provides first human data indicating that mGluR2/3agonism is involved in autonomic responsiveness, suggesting an important role of mGluR2/3in central autonomic regulation.

In cancer, proto-oncogenes are often altered by genomic amplification.  Here we report recurrent focal amplifications of chromosomal segment 4q12 overlapping the proto-oncogenes PDGFRA and KIT in non-small cell lung cancer (NSCLC).  Single nucleotide polymorphism (SNP) array and fluorescent in situ hybridization (FISH) analysis indicate that 4q12 is amplified in 3-7% of lung adenocarcinomas and 8-10% of lung squamous cell carcinomas.  In addition, we demonstrate that the NSCLC cell line NCI-H1703 exhibits focal amplification of PDGFRA and is dependent on PDGFRα activity for cell growth.  Treatment of NCI-H1703 cells with PDGFRA-specific shRNAs or with the PDGFRα/KIT small molecule inhibitors imatinib or sunitinib leads to cell growth inhibition. However, these observations do not extend to NSCLC cell lines with lower-amplitude and broader gains of chromosome 4q.  Together these observations implicate PDGFRA and KIT as potential oncogenes in NSCLC, but further study is needed to define the specific characteristics of those tumors that could respond to PDGFRα/KIT inhibitors.

The effects of 5-hydroxytryptamine 1A (5-HT(1A)) receptor ligands on aversive learning were examined in the passive avoidance (PA) task in mice. Anxiety and autonomic functions were investigated using the elevated plus-maze and heart rate measurements. The main findings from this study are as follows. 1) Pretraining administration of the 5-HT(1A) receptor agonist 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide] facilitated PA retention at low doses (0.01 and 0.03 mg/kg) but impaired PA retention at higher doses (0.1-1.0 mg/kg), consistent with previous findings in the rat. 2) Similar to the acetylcholinesterase inhibitor physostigmine, pretraining administration of the 5-HT(1A) receptor antagonists [(R)-3-N,N-dicyclobutylamino-8 fluoro-3,4-dihydro-3H-1-benzopyran-5-carboxamide hydrogen(2R,3R)-tartrate monohydrate] NAD-299 (0.1-2 mg/kg) and [N-2-4-(2-methoxyphenyl)-1-piperazinylethyl-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride] WAY-100635 (0.3-3 mg/kg) enhanced PA retention. 3) The impairment (1 mg/kg) but not the facilitation (0.03 mg/kg) induced by 8-OH-DPAT was fully blocked by NAD-299 (0.3 mg/kg). 4) 5-HT(1A) receptor ligands given immediate post-training failed to alter PA retention. 5) NAD-299 (0.3-1 mg/kg) blocked the impairment of PA retention caused by a) the nonselective muscarinic receptor antagonist scopolamine and b) the non-competitive N-methyl-d-aspartate receptor antagonist MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate]. 6) A subthreshold dose of scopolamine completely blocked the facilitatory effect of NAD-299 on PA retention. 7) Anxiety-related behaviors and autonomic function were unchanged by NAD-299. 8) In situ hybridization showed that septal neurons expressing 5-HT(1A) receptor mRNA were codistributed with markers for cholinergic, GABAergic, and glutamatergic neurons. These results indicate that systemic administration of 5-HT(1A) receptor antagonists can facilitate cognitive performance, most likely by enhancing hippocampal/cortical cholinergic and glutamatergic neurotransmissions. Selective 5-HT(1A) receptor antagonists may be useful in the treatment of cognitive deficits such as Alzheimer's disease.

Although there are powerful drugs against infectious diseases and cancer on the market, delivery systems are needed to decrease serious toxic and noncurative side effects. In order to enhance compliance, several delivery systems such as polymeric micro- and nanoparticles, liposomal systems and erythrocyte ghosts have been developed. Bacterial ghosts representing novel advanced delivery and targeting vehicles suitable for the delivery of hydrophobic or water-soluble drugs, are the main focus of this review. They are useful nonliving carriers, as they can carry different active substances in more than one cellular location separately and simultaneously. Bacterial ghosts combine excellent natural or engineered adhesion properties with versatile carrier functions for drugs, proteins and DNA plasmids or DNA minicircles. The simplicity of both bacterial ghost production and packaging of drugs and/or DNA makes them particularly suitable for the use as a delivery system. Further advantages of bacterial ghost delivery vehicles include high bioavailability and a long shelf life without the need of cold-chain storage due to the possibility to freeze-dry the material.

The actions of the neuropeptide corticotropin-releasing factor (CRF) are modulated by a CRF binding protein (CRFBP). In view of the memory-enhancing effects of CRF, the release of endogenous CRF from CRFBP by CRFBP inhibitors has been suggested as a therapeutical strategy for the treatment of cognitive deficits. This mini-review will summarize recent advances in the field with a focus on the pharmaceutical potential of CRFBP inhibitors.

The present study was performed to 1) determine heart rate (HR) effects mediated through central corticotropin-releasing factor receptor subtypes 1 (CRF(1)) investigate and 2 (CRF(2)) and 2) to the contribution of endogenous CRF to baseline HR and its fear-induced adjustment in freely moving mice. CRF ligands were injected into both lateral ventricles (i.c.v.) 15 min before the presentation of a conditioned auditory fear stimulus (CS). Initial behavioral results suggest an ovine CRF (oCRF)-mediated enhanced baseline fear and mildly enhanced conditioned auditory fear. In contrast, i.c.v. injection of oCRF (35-210 ng/mouse) dose-dependently decreased baseline HR, increased HR variability, and attenuated the CS-induced tachycardia. This effect is suggested to depend on a combined activation of sympathetic and parasympathetic activity referred to as enhanced sympathovagal antagonism. An extreme bradycardia was elicited by oCRF injection into the lower brainstem. All HR effects were probably mediated by CRF(1) because injection of the CRF(2)-selective agonist mouse urocortin II was ineffective, and the baseline bradycardia by i.c.v. CRF was preserved in CRF(2)-deficient mice. Injection of various CRF receptor antagonists including the CRF(2)-selective antisauvagine-30 did not affect the conditioned HR response. This finding suggests that endogenous CRF does not contribute to the fear-mediated tachycardia. Thus, the hypothesis of an involvement of CRF in HR responses of mice to acute aversive stimulation is rejected. Pharmacological evidence points at the involvement of CRF(1) in enhanced sympathovagal antagonism, a pathological state contributing to elevated cardiac risk, whereas the physiological role of the brain CRF system in cardiovascular regulation remains to be determined.

The action of corticotropin-releasing factor (CRF) is mediated by two recently identified receptors, CRFR1 and CRFR2, that differ with respect to their anatomical distribution and pharmacologic ligand-binding properties. Here we show by an analysis of circadian heartbeat interval fluctuations that CRFR2-deficiency in mice does not interfere with the dynamical mechanisms underlying the control of heart rate. Hence, intact CRFR2 would not constitute an indispensable requirement of physiologic cardiac rhythm regulation. However, both CRFR2 knockout (−/−) and wildtype control (+/+) mice showed altered dynamical properties of cardiac interbeat fluctuations in contrast to homogenetic inbred strains of mice (C57BL/6N and C57BL/6J). The results stress the impact of genetic background and support the generalized notion that transgenic 129/Sv-derived knockout mice exhibit altered cardiac dynamics which is interpreted to reflect an attenuation of neuroautonomic sympatho-vagal antagonism.

The omega-neuron 2 (ON2) is an auditory interneuron in Acheta domesticus that could play an important role in prothoracic auditory information processing. ON2 neurons were intracellularly recorded and iontophoretically stained with lucifer-yellow. The morphology of ON2 is similar to that described in other cricket species. Wholemounts and transverse sections of prothoracic ganglia show that ON2 branches cover the same regions as the omega-neuron 1 (ON1) branches except that ON2 has crossover branches in the anterior ring tract from both the ipsilateral and contralateral side. Auditory responses of ON2 are measured with spiking thresholds of about 60 dB SPL at 16 kHz (courtship song frequency) and about 80 dB SPL at 5 kHz (calling song frequency) under open field and closed field (monaural) stimulation. Open and closed field stimulations show almost identical threshold curves and response magnitudes to soma-ipsilateral and soma-contralateral stimulation. These data do not support a function of ON2 for directional hearing. In addition to excitation, ON2 receives inhibitory inputs from both ears at 5 and 16 kHz. Subthreshold inhibition is obvious in responses with open and closed field stimulation at 5 kHz. Responses show mixed excitation and inhibition at intensities above 80 dB SPL at 5 and 16 kHz. These data confirm previous immunohistochemical results showing GABAergic input to ON2 in Gryllus bimaculatus. Two newly described local neurons (“LN1”, “LN2”), which are probably nonspiking and have low 5-kHz thresholds, could be candidates for the inhibitory input to ON2. J. Exp. Zool. 279:43-53, 1997. © 1997 Wiley-Liss, Inc.

ABSTRACTThe syllables of the calling songs of many bushcrickets (Orthoptera, Tettigoniidae) are formed of a series of discrete sound impulses. Bioacoustic investigations of E. ephippigerrevealed that in some males the number of impulses per main syllable fall from about 50 impulses down to 10 impulses with increasing age of the male after the final moult. An influence of the altered signal on the frequency content of the acoustic signal could not be proved as far as averaged spectra are concerned. The geriatric effect on the syllable pattern is caused by mechanical attrition of the peaks of the teeth forming the pars stridens.Two-choice experiments revealed that females can discriminate between songs with modified impulse repetition rate and songs with the normal impulse repetition rate. The inter-impulse intervals in the modified song vary from 0.8 ms up to 8.7 ms. Experiments with stimuli formed of natural impulses with a constant repetition rate proved that females are able to discriminate between these signals over a certain range. A stimulus that contains an inter-impulse interval similar to the species-specific one (1.87 ms on average) is preferred to stimuli with altered (∼ > ± 1ms) inter-impulse interval when broadcast alternately. When the impulse repetition rate is only slightly altered (∼ < ± 1ms) the females cannot discriminate between these signals behaviourally.

Investigations on the locomotor behaviour of females of the bushcricketEphippiger ephippigerwere carried out. Their behaviour was observed on a multibranched plant in the laboratory while both conspecific song and a simultaneously transmitted vibratory signal were applied to the plant. As soon as the insects reached a certain level in the bush the acoustic stimulus was switched off and only vibratory cues (tremulation) remained. Observations were then made of females using these vibratory signals to orientate towards the source of the vibratory signals (artificial male). The females were attracted at short distance (58 cm) only. At a longer distance (105 cm) the insects did not show definite vibrotaxis. A comparison which was made to an experiment with conspecific song and simultaneously transmitted vibration revealed that females were much more attracted by these combined stimuli. Here the females made a lower number of interruptions and reached the emitter faster than if stimulated with tremulation alone.Investigations on the locomotor behaviour of females of the bushcricketEphippiger ephippigerwere carried out. Their behaviour was observed on a multibranched plant in the laboratory while both conspecific song and a simultaneously transmitted vibratory signal were applied to the plant. As soon as the insects reached a certain level in the bush the acoustic stimulus was switched off and only vibratory cues (tremulation) remained. Observations were then made of females using these vibratory signals to orientate towards the source of the vibratory signals (artificial male). The females were attracted at short distance (58 cm) only. At a longer distance (105 cm) the insects did not show definite vibrotaxis. A comparison which was made to an experiment with conspecific song and simultaneously transmitted vibration revealed that females were much more attracted by these combined stimuli. Here the females made a lower number of interruptions and reached the emitter faster than if stimulated with tremulation alone.

K-RAS–mutated lung adenocarcinomas depend on ERBB signaling, and pan-ERBB inhibitors impair K-RAS–driven lung tumorigenesis.

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