Distinct cellular pools of perilipin 5 point to roles in lipid trafficking.

The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~1.15 g/ml) and size (~575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.

Morphology of the Vasculature and Blood Supply of the Brown Adipose Tissue Examined in an Animal Model by Micro-CT.

We performed micro-CT imaging of the vascular blood supply in the interscapular area of the brown adipose tissue in three mice with the use of intravascular contrast agent Aurovist™. Resulting 3D data rendering was then adapted into 2D resolution with visualization using false color system and grayscale images. These were then studied for the automatic quantification of the blood vessel density within this area. We found the highest most occurring density within arterioles or venules representing smaller blood vessels whereas with the increase of the vessel diameters a lower percentage rate of their presence was observed in the sample. Our study shows that micro-CT scanning in combination with Aurovist™ contrast is suitable for anatomical studies of interscapular area of brown adipose tissue blood vessel supply.

Easy Identification of Optimal Coronal Slice on Brain Magnetic Resonance Imaging to Measure Hippocampal Area in Alzheimer's Disease Patients.

INTRODUCTION: Measurement of an- hippocampal area or volume is useful in clinical practice as a supportive aid for diagnosis of Alzheimer's disease. Since it is time-consuming and not simple, it is not being used very often. We present a simplified protocol for hippocampal atrophy evaluation based on a single optimal slice in Alzheimer's disease. METHODS: We defined a single optimal slice for hippocampal measurement on brain magnetic resonance imaging (MRI) at the plane where the amygdala disappears and only the hippocampus is present. We compared an absolute area and volume of the hippocampus on this optimal slice between 40 patients with Alzheimer disease and 40 age-, education- and gender-mateched elderly controls. Furthermore, we compared these results with those relative to the size of the brain or the skull: the area of the optimal slice normalized to the area of the brain at anterior commissure and the volume of the hippocampus normalized to the total intracranial volume. RESULTS: Hippocampal areas on the single optimal slice and hippocampal volumes on the left and right in the control group were significantly higher than those in the AD group. Normalized hippocampal areas and volumes on the left and right in the control group were significantly higher compared to the AD group. Absolute hippocampal areas and volumes did not significantly differ from corresponding normalized hippocampal areas as well as normalized hippocampal volumes using comparisons of areas under the receiver operating characteristic curves. CONCLUSION: The hippocampal area on the well-defined optimal slice of brain MRI can reliably substitute a complicated measurement of the hippocampal volume. Surprisingly, brain or skull normalization of these variables does not add any incremental differentiation between Alzheimer disease patients and controls or give better results.

Asymmetric Changes in Limbic Cortex and Planum Temporale in Patients with Alzheimer Disease.

BACKGROUND: There are several cortical areas related to the limbic system that form the output from the hippocampal formation whose cellular and morphological features are important for the onset and progression of AD. We hypothesized that there would be a significant difference in the size of cortical pyramidal neurons and that there would also be a hemispheric asymmetry between Alzheimer disease patients and controls. These differences would potentially be accompanied by an increase in the numbers of Fluoro-Jade B-positive degenerating cortical neurons and a corresponding decrease in the numbers of DAPI-stained cortical neuronal nuclei in subjects with AD compared to controls. Such changes could potentially be used as another marker in postmortem neuropathological diagnosis of AD. METHODS: We measured absolute numbers of DAPI and Fluoro-Jade B stained cells in five cortical areas of the limbic system and four subareas of planum temporale in the post-mortem brains of subjects with Alzheimer disease. We also measured the size of pyramidal neurons in layer III in the five cortical areas of the limbic system in these subjects. All measurements were performed separately for the left and right hemisphere in order to identify asymmetries between the two hemispheres. RESULTS: We observed a significant decrease in numbers of DAPI stained cells in layers IV-VI of the anterior cingulate gyrus on the right side, in layers I-III of the posterior cingulate gyrus on the left side, in layers IV-VI in the transition region from superior temporal gyrus into planum temporale on the right and in layers IV-VI in the transition from planum temporale to insular cortex on the left. We also observed a significant increase in the numbers of Fluoro-Jade stained cells in layers I-III of the anterior cingulate gyrus and in layers I-III on the left and layers IV-VI of the right gyrus of Heschl. Shortening of the size of layer III pyramidal neurons in subjects with Alzheimer´s disease was found in the anterior cingulate gyrus on the right, in the posterior cingulate gyrus and entorhinal cortex on the left and on the right in the parahippocampal gyrus. CONCLUSION: Our study demonstrates asymmetries in different cortical regions of the temporal lobe that can be used as another marker in the postmortem diagnosis of AD.

De novo design of antibody complementarity determining regions binding a FLAG tetra-peptide.

Computational antibody engineering efforts to date have focused on improving binding affinities or biophysical characteristics. De novo design of antibodies binding specific epitopes could greatly accelerate discovery of therapeutics as compared to conventional immunization or synthetic library selection strategies. Here, we employed de novo complementarity determining region (CDR) design to engineer targeted antibody-antigen interactions using previously described in silico methods. CDRs predicted to bind the minimal FLAG peptide (Asp-Tyr-Lys-Asp) were grafted onto a single-chain variable fragment (scFv) acceptor framework. Fifty scFvs comprised of designed heavy and light or just heavy chain CDRs were synthesized and screened for peptide binding by phage ELISA. Roughly half of the designs resulted in detectable scFv expression. Four antibodies, designed entirely in silico, bound the minimal FLAG sequence with high specificity and sensitivity. When reformatted as soluble antigen-binding fragments (Fab), these clones expressed well, were predominantly monomeric and retained peptide specificity. In both formats, the antibodies bind the peptide only when present at the amino-terminus of a carrier protein and even conservative peptide amino acid substitutions resulted in a complete loss of binding. These results support in silico CDR design of antibody specificity as an emerging antibody engineering strategy.

Altered levels of urokinase on monocytes and in serum of children with AIDS; effects on lymphocyte activation and surface marker expression.

Urokinase (UK) type plasminogen activator is a serine protease produced by activated human monocytes. Despite the well-documented roles played by UK in cell-mediated immunity in healthy humans, the roles played by UK in the derangements of cell-mediated immune responses observed in HIV disease remain largely undefined. In these studies the numbers of peripheral blood lymphocytes and monocytes bearing surface UK (UK+) as well as serum levels of UK (flow microfluorimetry and ELISA, respectively) were determined in children with AIDS and in healthy HIV-negative children. The effects of exogenous UK on lymphocyte activation (cell cycle analysis using living cells) and surface marker (CD3, CD4, CD8, and CD19) expression (flow microfluorimetry using fixed cells) were also studied. Data are expressed as percent total cells. Numbers of UK+ lymphocytes in children with AIDS were similar to those observed in healthy children. In contrast, numbers of UK+ peripheral blood monocytes were dramatically decreased (> 70%) in the children with AIDS. However, serum levels of UK were increased (nearly threefold) in these children. When lymphocytes from these children were cultured with soluble UK, numbers of cells in S phase of cell cycle appeared suppressed. Incubation of fixed lymphocytes from either a child with AIDS or from a healthy child with exogenous UK appeared to increase numbers of cells expressing CD3. Incubation with UK had no effect on expression of any other surface marker (CD4, CD8, or CD19) using cells from the child with AIDS. In contrast, incubation with UK appeared to decrease (fivefold) numbers of cells expressing CD19 and increase numbers of cells expressing CD4 and CD8 only when fixed lymphocytes from a healthy HIV-negative child were used. The results suggest important roles for UK in regulation of lymphocyte surface markers in general and in CD3- and CD19-dependent lymphocyte activation pathways specifically. Furthermore, these studies add to a widening body of evidence implicating UK dysregulation in the pathogenesis of HIV disease and may point to pharmacological opportunities involving UK to delay or prevent progression of HIV infection into full-blown AIDS.

Experimental lesion of medial frontal cortex mediates inhibition of expression of carbohydrate-binding sites in the spleen macrophages in rat.

The fundamental concept of a neuroimmunological network is well appreciated although detailed description of the individual mechanisms has not yet been attained. In an effort to close this gap, the effect of electrolesion of the frontal medial cortex, a structure with the known "immunoreactivity", on selected phenotypic features of spleen macrophages was studied. Since sugar receptors (lectins) are pivotal for recognition, custom-made tools termed neoglycoproteins were employed to delineate any injury-induced changes of their expression. The total number of macrophages in the spleen red pulp was assessed using the ED-1 monoclonal antibody. The results showed that after lesion of the medial frontal cortex, the extent of expression of carbohydrate-binding sites in red pulp spleen macrophages significantly decreased without affecting the total number of these cells. These data intimate that distinct brain regions are involved in the control of the phenotype of macrophages in the central lymphoid organs by currently elusive biochemical mechanisms.

Delayed effects of elevated corticosterone level on volume of hippocampal formation in laboratory rat.

We studied delayed effects of elevated plasma levels of corticosterone (Cort) on volumetry, neuronal quantity, and gross marks of neurodegeneration in the hippocampal formation of Long-Evans rats. Animals were exposed to increased CORT levels for three weeks via implanted subcutaneous pellets. Volumetry, neuronal quantification and gross marks of degeneration were measured seven weeks after the termination of CORT treatment. We observed significant differences in volumes and especially in laterality of hippocampal subfields between control and CORT-treated animals. We found that the left hippocampus was substantially larger than the right hippocampus in the corticosterone-treated group, but not in the control group. In the control group, on the other hand, right hippocampal volume was markedly higher than all other measured volumes (hippocampal left control, hippocampal left CORT-treated and hippocampal right CORT-treated). Left hippocampal volume did not differ between the groups.

Sex-dependent actions of amyloid beta peptides on hippocampal choline carriers of postnatal rats.

It is suggested that amyloid beta peptides (Abeta) play a role in the pathogenesis of Alzheimer disease but their physiological function is still unknown. However, low pM-nM concentrations mediate a hypofunction of a basal forebrain cholinergic system without marked signs of neurotoxicity. In this study, we compared in vitro effects of soluble nonaggregated human Abeta 1-40 and 1-42 either on synaptosomal hemicholinium-3 sensitive choline carriers or on membrane fluidity in hippocampi of male and female Wistar rats aged 7 and 14 days or 2-3 months. The results indicate age- and sex-dependent effects mediated by peptides at nM concentrations but no significant differences between both fragments. Namely, opposite actions were observed in 14-day (the increase in the choline uptake and membrane fluidity) when compared to 7-day old and adult males (the mild drops). Lineweaver-Burk plot analysis revealed that the enhancement of the high-affinity choline transport in 14-day old males occurs via alterations in K (M )and the change was accompanied by a mild increase in the specific binding of [3H]hemicholinium-3. On the other hand, no age-dependent differences were found in females. Rat Abeta 1-40 mediated similar effects on 14-day old rats as the corresponding human fragment. Moreover, higher levels of soluble peptides were detected in immature when compared to mature male brains by means of competitive ELISA. Our study indicates that Abeta could play a role in postnatal sexual differentiation of hippocampal cholinergic system.

Exposure of postnatal rats to a static magnetic field of 0.14 T influences functional laterality of the hippocampal high-affinity choline uptake system in adulthood; in vitro test with magnetic nanoparticles.

Our previous experiments indicated an age- and sex-dependent functional lateralization of a high-affinity choline uptake system in hippocampi of Wistar rats. The system is connected with acetylcholine synthesis and also plays a role in spatial navigation. The current study demonstrates that a single in vivo exposure of 7- or 14-day-old males to a static magnetic field of 0.14 T for 60-120 min evokes asymmetric alterations in the activity of carriers in adulthood. Namely, the negative field (antiparallel orientation with a vertical component of the geomagnetic field) mediated a more marked decrease in the right hippocampus. The positive field (parallel orientation) was ineffective. Moreover, differences between the carriers from the right and the left hippocampi were observed on synaptosomes pretreated with superparamagnetic nanoparticles and exposed for 30 min in vitro. The positive field enhanced more markedly the activity of carriers from the right hippocampus, the negative that from the left hippocampus, on the contrary. Our results demonstrate functionally teratogenic risks of the alterations in the orientation of the strong static magnetic field for postnatal brain development and suggest functional specialization of both hippocampi in rats. Choline carriers could be involved as secondary receptors in magnetoreception through direct effects of geomagnetic field on intracellular magnetite crystals and nanoparticles applied in vivo should be a useful tool to evaluate magnetoreception in future research.

The brain lesion influences the numbers of peripheral blood leukocytes in the rat.

The mutual relationship between the central nervous and immune system are intensively studied. The lesion of distinct structures of the rat brain such as septum influence the model immune response such as lymphocyte proliferation and delayed skin hypersensitivity. Employing the model of the damage of septum in the rat brain by electrolesion we demonstrated the decrease of the number of peripheral blood leukocytes, mainly cells exhibiting CD25 and CD45RA antigens in the rat. The striatum destruction has much lower influence on the studied parameters, which suggests a specific effect of the septum on these hematological parameters.

Brain lesion-induced alteration of selected phenotypic properties of spleen macrophages and their partial restoration in the course of foreign body reaction against intraperitoneally implanted polymers.

A lesion in the dorsoposterior part of the rat brain septum is known to exert an inhibitory effect on the delayed skin hypersensitivity and incorporation of radiolabeled thymidine into the lymphoid organs. To determine whether distinct properties of macrophages will also be modulated by this type of injury, we have focused upon the monitoring of expression of sugar receptors (lectins). In this study we show a reduction in the number of macrophages expressing carbohydrate-binding sites for asialoglycoproteins (beta-D-galactoside), alpha-D-mannoside and alpha-D-mannoside-6-phosphate in spleen macrophages after the lesion of the dorsoposterior septum of the brain in the rat. The number of ED-1+ macrophages was not influenced. The intraperitoneal injection of beads prepared from the copolymer of 2-hydroxyethyl methacrylate with dimethyl aminoethyl methacrylate (30 wt %) elevated significantly the number of ED-1+ spleen macrophages and number of macrophages with binding site(s) recognizing asialoglycoproteins and alpha-D-mannoside-6-phosphate, respectively. These results indicate that a foreign-body reaction appears to be able to mediate a phenotypic restoration of lectin expression by spleen macrophages altered by the brain lesion. It can be suggested that, for example, a probable production of cytokines by the inflammatory cells colonizing the implanted beads plays a role in this process.