Resonance Raman spectroscopy of xanthophylls in pigment mutant thylakoid membranes of pea

Low-temperature resonance Raman spectroscopy was used to study the changes in the molecular structure and configuration of the major xanthophylls in thylakoid membranes isolated from mutants of pea with modified pigment content and altered structural organization of their pigment-protein complexes. The Raman spectra contained four known groups of bands, nu(1)-nu(4), which could be assigned to originate mainly from the long wavelength absorbing lutein and neoxanthin upon 514.5 nm and at 488 nm excitations, respectively. The overall configuration of these bound xanthophyll molecules in the mutants appeared to be similar to the wild type, and the configuration in the wild type was almost identical with that in the isolated main chlorophyll a/b light harvesting protein complex of photosystem II (LHCII). Significant differences were found mainly in the region of nu(4) (around 960 cm(-1)), which suggest that the macroorganization of PS II-LHCII supercomplexes and/or of the LHCII-only domains are modified in the mutants compared to the wild type.     

Cytoplasmic sequestration of HDAC7 from mitochondrial and nuclear compartments upon initiation of apoptosis

Control of global histone acetylation status is largely governed by the opposing enzymatic activities of histone acetyltransferases and deacetylases (HDACs). HDACs were originally identified as modulators of nuclear histone acetylation status and have been linked to chromosomal condensation and subsequent gene repression. Accumulating evidence highlights HDAC modification of non-histone targets. Mitochondria were first characterized as intracellular organelles responsible for energy production through the coupling of oxidative phosphorylation to respiration. More recently, mitochondria have been implicated in programmed cell death whereby release of pro-apoptotic inner membrane space factors facilitates apoptotic progression. Here we describe the novel discovery that the nuclear encoded Class II human histone deacetylase HDAC7 localizes to the mitochondrial inner membrane space of prostate epithelial cells and exhibits cytoplasmic relocalization in response to initiation of the apoptotic cascade. These results highlight a previously unrecognized link between HDACs, mitochondria, and programmed cell death.     

Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis

Spatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol-anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide-differentiated HL60 cells in a pertussis toxin-sensitive, actin-dependent manner. A transmembrane, nonraft GFP protein was distributed homogeneously in moving cells. A GFP-CCR5 chimera, which partitions in L rafts, accumulated at the leading edge, and CCR5 redistribution coincided with recruitment and activation of phosphatidylinositol-3 kinase gamma in L rafts in polarized, moving cells. Membrane cholesterol depletion impeded raft redistribution and asymmetric recruitment of PI3K to the cell side facing the chemoattractant source. This is the first direct evidence that lipid rafts order spatial signaling in moving mammalian cells, by concentrating the gradient sensing machinery at the leading edge.     

Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1

Receptor oligomerization is vital for activating intracellular signaling, in part by initiating events that recruit effector and adaptor proteins to sites of active signaling. Whether these distal molecules themselves oligomerize is not well appreciated. In this study, we examined the molecular interactions of the adaptor protein GRB2. In T cells, the SH2 domain of GRB2 binds phosphorylated tyrosines on the adaptor protein LAT and the GRB2 SH3 domains associate with the proline-rich regions of SOS1 and CBL. Using biochemical and biophysical techniques in conjunction with confocal microscopy, we observed that the simultaneous association of GRB2, via its SH2 and SH3 domains, with multivalent ligands led to the oligomerization of these ligands, which affected signaling. These data suggest that multipoint binding of distal adaptor proteins mediates the formation of oligomeric signaling clusters vital for intracellular signaling.     

Quality of life of patients after stroke in county Osijek-Baranya

The purpose of this prospective study was to determine quality of life of patients after stroke in Osijek-Baranya County. The research included 161 patients (82 men and 79 women) who had their first acute stroke and were treated at Department of Neurology, Osijek University Hospital Center The Barthel Index (BI) was used to assess functional deficiency and SS-QOL (Stroke-Specific Quality of Life) questionnaire was used for self-evaluation of patients' physical and mental health. The first assessment was carried out in the acute phase of the disease, and control assessments 30, 90 and 180 days after the stroke. Mean Barthel Index score was higher at every successive measurement (55, 80, 95, 95). All BI items were statistically significant (Friedman, p < 0.001) apart from dressing and bowel control. BI score indicated greater dependence in women in all assessments except for those taken 90 days after onset of symptoms (chi2-test, p = 0.111). Mean values of SS-QOL for physical health were: 105.2, 98.3, 105.7, 117.5 and for mental health: 64.24, 57.9, 64.3, 68.1. Statistically significant difference was present in men, both for physical health (Friedman p = 0.009) and total SS-QOL (Friedman p = 0.014), while in women there was no statistically significant difference between the measurements (Friedman p = 0.719). The research showed that stroke has significant influence on basic and specific daily life activities and interferes with the quality of life of stroke patients. Women have lower level of independence. Patients who live with their families make better evaluation of their physical and mental health.     

Inhibitory effect of LXR activation on cell proliferation and cell cycle progression through lipogenic activity

Liver X receptor (LXR), a sterol-activated nuclear hormone receptor, has been implicated in cholesterol and fatty acid homeostasis via regulation of reverse cholesterol transport and de novo fatty acid synthesis. LXR is also involved in immune responses, including anti-inflammatory action and T cell proliferation. In this study, we demonstrated that activated LXR suppresses cell cycle progression and proliferation in certain cell types. Stimulation of LXR with synthetic ligand T0901317 or GW3965 inhibited cell growth rate and arrested the cell cycle at the G1/S boundary in several cells, such as RWPE1, THP1, SNU16, LNCaP, and HepG2. However, LXR ligands did not exhibit antiproliferative activity in PC3, HEK293, or HeLa cells. Interestingly, activated LXR-mediated cell cycle arrest is closely correlated with the lipogenic gene expression and triacylglyceride accumulation. In accordance with these findings, suppression of FAS via small-interference RNA (siRNA) partially alleviated the antiproliferative effect of LXR activation in RWPE1 cells. Together, these data suggest that LXR activation with its ligands inhibits cell proliferation and induces G1/S arrest through elevated lipogenic activity, thus proposing a novel effect of activated LXR on cell cycle regulation.     

Peripheral blood neutrophil activation patterns are associated with pulmonary inflammatory responses to lipopolysaccharide in humans

Increased nuclear accumulation of NF-kappaB in LPS-stimulated peripheral blood neutrophils has been shown to be associated with more severe clinical course in patients with infection associated acute lung injury. Such observations suggest that differences in neutrophil response may contribute to the pulmonary inflammation induced by bacterial infection. To examine this question, we sequentially measured LPS-induced DNA binding of NF-kappaB in neutrophils collected from healthy humans on at least three occasions, each separated by at least 2 wk, and then determined pulmonary inflammatory responses after instillation of LPS into the lungs. Consistent patterns of peripheral blood neutrophil responses, as determined by LPS-induced NF-kappaB DNA binding, were present in volunteers, with a >80-fold difference between individuals in the mean area under the curve for NF-kappaB activation. The number of neutrophils recovered from bronchoalveolar lavage after exposure to pulmonary LPS was significantly correlated with NF-kappaB activation in peripheral blood neutrophils obtained over the pre-LPS exposure period (r = 0.65, p = 0.009). DNA binding of NF-kappaB in pulmonary neutrophils also was associated with the mean NF-kappaB area under the curve for LPS-stimulated peripheral blood neutrophils (r = 0.63, p = 0.01). Bronchoalveolar lavage levels of IL-6 and TNFRII were significantly correlated with peripheral blood neutrophil activation patterns (r = 0.75, p = 0.001 for IL-6; and r = 0.48, p = 0.049 for TNFRII. These results demonstrate that stable patterns in the response of peripheral blood neutrophils to LPS exist in the human population and correlate with inflammatory response following direct exposure to LPS in the lung.     

Recruitment and activation of PLCgamma1 in T cells: a new insight into old domains

Engagement of the T-cell antigen receptor leads to recruitment of phospholipase Cgamma1 (PLCgamma1) to the LAT-nucleated signaling complex and to PLCgamma1 activation in a tyrosine phosphorylation-dependent manner. The mechanism of PLCgamma1 recruitment and the role of PLCgamma1 Src homology (SH) domains in this process remain incompletely understood. Using a combination of biochemical methods and real-time fluorescent imaging, we show here that the N-terminal SH2 domain of PLCgamma1 is necessary but not sufficient for its recruitment. Either the SH3 or C-terminal SH2 domain of PLCgamma1, with the participation of Vav1, c-Cbl and Slp76, are required to stabilize PLCgamma1 recruitment. All three PLCgamma1 SH domains are required for phosphorylation of PLCgamma1 Y783, which is critical for enzyme activation. These novel findings entailed revision of the currently accepted model of PLCgamma1 recruitment and activation in T lymphocytes.     

Genome rearrangements, deletions, and amplifications in the natural population of Bartonella henselae

Cats are the natural host for Bartonella henselae, an opportunistic human pathogen and the agent of cat scratch disease. Here, we have analyzed the natural variation in gene content and genome structure of 38 Bartonella henselae strains isolated from cats and humans by comparative genome hybridizations to microarrays and probe hybridizations to pulsed-field gel electrophoresis (PFGE) blots. The variation in gene content was modest and confined to the prophage and the genomic islands, whereas the PFGE analyses indicated extensive rearrangements across the terminus of replication with breakpoints in areas of the genomic islands. We observed no difference in gene content or structure between feline and human strains. Rather, the results suggest multiple sources of human infection from feline B. henselae strains of diverse genotypes. Additionally, the microarray hybridizations revealed DNA amplification in some strains in the so-called chromosome II-like region. The amplified segments were centered at a position corresponding to a putative phage replication initiation site and increased in size with the duration of cultivation. We hypothesize that the variable gene pool in the B. henselae population plays an important role in the establishment of long-term persistent infection in the natural host by promoting antigenic variation and escape from the host immune response.