Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging

Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein-protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein-protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.     

Mice with neonatally induced inactivation of the vascular cell adhesion molecule-1 fail to control the parasite in Toxoplasma encephalitis

Under various inflammatory conditions, cell adhesion molecules are up-regulated in the central nervous system (CNS) and may contribute to the recruitment of leukocytes to the brain. In the present study, the functional role of vascular cell adhesion molecule (VCAM)-1 in Toxoplasma encephalitis (TE) was addressed using VCAM(flox/flox MxCre) mice. Neonatal inactivation of the VCAM-1 gene resulted in a lack of induction of VCAM-1 on cerebral blood vessel endothelial cells, whereas the constitutive expression of VCAM-1 on choroid plexus epithelial cells and the ependyma was unaffected; in these animals, resistance to T. gondii was abolished, and VCAM(flox/flox MxCre) mice died of chronic TE caused by a failure to control parasites in the CNS. Although leukocyte recruitment to the CNS was unimpaired, the B cell response was significantly reduced as evidenced by reduced serum levels of anti-T. gondii-specific IgM and IgG antibodies. Furthermore, the frequency and activation state of intracerebral T. gondii-specific T cells were decreased, and microglial activation was markedly reduced. Taken together, these data demonstrate the crucial requirement of VCAM-1-mediated immune reactions for the control of an intracerebral infectious pathogen, whereas other cell adhesion molecules can efficiently compensate for VCAM-1-mediated homing across cerebral blood vessels.     

Responses to natural scenes in cat V1

Studies on processing in primary visual areas often use artificial stimuli such as bars or gratings. As a result, little is known about the properties of activity patterns for the natural stimuli processed by the visual system on a daily basis. Furthermore, in the cat, a well-studied model system for visual processing, most results are obtained from anesthetized subjects and little is known about neuronal activations in the alert animal. Addressing these issues, we measure local field potentials (lfp) and multiunit spikes in the primary visual cortex of awake cats. We compare changes in the lfp power spectra and multiunit firing rates for natural movies, movies with modified spatio-temporal correlations as well as gratings. The activity patterns elicited by drifting gratings are qualitatively and quantitatively different from those elicited by natural stimuli and this difference arises from both spatial as well as temporal properties of the stimuli. Furthermore, both local field potentials and multiunit firing rates are most sensitive to the second-order statistics of the stimuli and not to their higher-order properties. Finally, responses to natural movies show a large variability over time because of activity fluctuations induced by rapid stimulus motion. We show that these fluctuations are not dependent on the detailed spatial properties of the stimuli but depend on their temporal jitter. These fluctuations are important characteristics of visual activity under natural conditions and impose limitations on the readout of possible differences in mean activity levels.     

Back extensor muscle oxygenation and fatigability in healthy subjects and low back pain patients during dynamic back extension exertion

The purpose of this study was to assess if chronic low back pain patients have impaired paraspinal muscle O₂ turnover and endurance capacity as compared to healthy control subjects during dynamic exercise. Middle-aged healthy male subjects (n = 12, control) and male patients with chronic low back pain (n = 17, CLBP) participated in the study. L4–L5 level paraspinal muscle fatigue was objectively assessed during earlier validated 90 s dynamic back endurance test (spectral EMG, MPF_slope). Also EMG amplitude (EMG_amplitude) and initial MPF (MPF_initial) were assessed from the initial 5 s of the endurance contraction. Simultaneously near infrared spectroscopy (NIRS) was used for quantitative measurement of local L4–L5 paraspinal muscle O₂ consumption. Subcutaneous tissue thickness (ATT) was measured from the EMG and NIRS recording sites. The results indicated that control and CLBP groups were compatible as regarding anthropometric variables, paraspinal muscle activation levels (EMG_amplitude), initial MPF (MPF_initial) and ATT. When the ATT was used as a covariate in the ANOVA analysis, CLBP group did not show significantly greater paraspinal muscle fatigability (right MPF_slope – 12.2 ± 10.7%/min, left right MPF_slope – 12.6 ± 13.3%/min) or O₂ consumption (right NIRS_slope – 52.8 ± 79.6 μM/l/s) as compared to healthy controls (right MPF_slope – 11.9 ± 7.6%/min, left MPF_slope – 12.7 ± 8.6%/min, right NIRS_slope – 53.7 ± 95.2 μM/l/s). As a conclusion, these CLBP male patients did not show any impaired rate of paraspinal muscle oxygen consumption or excessive paraspinal muscle fatigability during dynamic exercise as compared with healthy controls. Subcutaneous tissue thickness has a strong influence on the NIRS and EMG amplitude measurements and, if unchecked, it could result in the false interpretation of the results.     

Sclerotomal origin of smooth muscle cells in the wall of the avian dorsal aorta.

The dorsal aorta is the earliest formed intraembryonic blood vessel. It is composed of an inner lining consisting of endothelial cells and an outer wall consisting of smooth muscle cells (SMCs) and fibrocytes. Aortic SMCs have been suggested to arise from several developmental lineages. Cephalic neural crest provides SMCs of the proximal part of the aorta, and SMCs of the distal part are derived from the paraxial mesoderm. Here, we show by using quail-chick chimerization that in the avian embryo, SMCs in the wall of the dorsal aorta at trunk level arise from the sclerotome. Our findings indicate a two-step process of aortic wall formation. First, non-paraxial mesoderm-derived mural cells accumulate at the floor of the aorta. We refer to these cells as primary SMCs. Second, SMCs from the sclerotome are recruited to the roof and sides of the aorta, eventually replacing the primary SMCs in the aortic floor.     

DFNA54, a third locus for low-frequency hearing loss

Nonsyndromic hereditary hearing impairment (NSHHI) is a highly heterogeneous disorder with more than 90 loci mapped, of which nearly one-half of the responsible genes are identified. In dominant NSSHI hearing loss is typically biased towards the high frequencies while low-frequency hearing loss is unusual. Only two NSHHI loci, DFNA1 and DFNA6/14/38, are associated with predominantly low- frequency loss. We mapped the loci harboring the gene responsible for autosomal dominant low-frequency hearing loss in a multigenerational family. The pedigree of a Swiss family with low-frequency hearing loss was established. Using genomic DNA, DFNA1 and DFNA6/14/38 were excluded by linkage analysis or by direct sequencing of the responsible gene. Genome-wide linkage analysis was performed using commercially available microsatellite markers. Two-point linkage analysis demonstrated linkage to chromosome 5q31, the locus for DFNA15, with a lod score of 6.32 at recombination fraction =0 for marker D5S436. Critical recombinations were seen at markers D5S1972 and D5S410. Sequencing of the corresponding gene POU4F3 yielded no pathogenic mutation segregating with the affected members. In addition to Wolfram syndrome gene 1 (DFNA6/14/38) and diaphanous (DFNA1) there is evidence for a third gene involved in low-frequency hearing loss located at DFNA15. Because of the differences in auditory phenotype and the absence of pathogenic mutation in the coding region of POU4F3 it is likely that there is a second gene in 5q31, designated DFNA54, associated with NSHHI.     

Fat in the intestine as a regulator of appetite--role of CCK

The present review summarizes the appetite-suppressing effects of intestinal fat in the regulation of food intake in humans, with a special focus on the role of cholecystokinin (CCK). Current evidence supports a role for intestinal fat (especially long-chain free fatty acids) acting via the peptide CCK as a physiological satiety pathway. The regulation of satiety is, however, complex and it is not surprising that multiple control systems exist. It is interesting to note that nutrients, such as hydrolysis products of fat in the small intestine, stimulate the release of satiety peptides, such as CCK or PYY, that serve as satiety signals. CCK, released from the gastrointestinal tract by the local action of digested food, exerts various functions: stimulation of gallbladder contraction and exocrine pancreatic secretion, inhibition of gastric emptying, and inhibition of appetite. CCK functions therefore (1) as a positive feedback signal to stimulate digestive processes and (2) as negative feedback signal to limit the amount of food consumed during an individual meal.     

Complement-mediated enhancement of HIV-1 neutralisation by anti-HLA antibodies derived from polytransfused patients

We have recently shown that 'alloimmune sera' derived from polytransfused patients (PTP sera) are able to recognise and neutralise HIV in vitro. In this study we try to identify the protein(s), which are recognised by the PTP sera and elucidate mechanisms responsible for the neutralising capacity of these sera. The PTP sera allowed immunoprecipitation (IP) of HLA class II molecules on HIV-infected cells. To detect a potential cross-reactivity of alloreactive antibodies (Ab) with the HIV envelope protein gp160 or its subunits gp120/gp41 and HLA proteins, ELISA and FACS analyses were performed. The lack of reactivity of the PTP sera against rsgp160 in ELISA or FACS analysis indicated that recognition of cells was independent of HIV infection. To clarify whether interaction of the PTP sera with target cells has any effect on the infection process, virus neutralisation assays were performed. Inhibition of HIV infection was observed only when virus was pre-incubated with the PTP sera. Complement enhanced neutralisation of HIV-1 significantly. This enhancement was not due to complement-mediated lysis, because pre-incubation of the target cells with PTP sera did not inhibit HIV replication. Therefore, the neutralising effect of the Ab was due to blocking of the viral attachment/fusion process and not to negative signalling after infection. Since steric hindrance is possible only when HLA and gp120/gp41 are in close vicinity, isolation of rafts and IP assays were performed. These experiments revealed that gp120 and MHC class II molecules are indeed co-localised. The close physical association of gp120/gp41 and HLA strongly supports a mechanism for neutralisation of HIV by anti-HLA-Ab based on steric hindrance.     

Detection of N-(3-oxohexanoyl)-L-homoserine lactone in mice infected with Yersinia enterocolitica serotype O8

Yersinia enterocolitica synthesizes N-acyl-L-homoserine lactone (AHL) signal molecules via the LuxR-LuxI homologues YenR-YenI. In this study we checked two prototypes of mouse-virulent Y. enterocolitica serotype O8 strains WA-314 and 8081 for AHL production in vitro and in vivo (mouse infection model). We used thin-layer chromatography in combination with the Escherichia coli AHL biosensor to identify the AHL species produced. We detected only OHHL [N-(3-oxohexanoyl)-L-homoserine lactone] and not HHL (N-hexanoyl-L-homoserine lactone) produced by Y. enterocolitica O8 in culture supernatant or infected mouse tissue. This is the first report demonstrating AHL production by yersiniae during infection.