Vascular endothelial growth factor receptor-2 and low affinity VEGF binding sites on human glomerular endothelial cells: Biological effects and advanced glycosilation end products modulation

Vascular Endothelial Growth Factor (VEGF), binding to its receptor in endothelial cells, seems to modulate the increased blood flow in the early phase of diabetic renal disease. The aim of the study was to evaluate, in a diabetic milieu, the expression, biological function and modulation of VEGF binding sites in human glomerular endothelial cells (GENC). We demonstrated the presence of VEGF binding sites with high (VEGFR-2) and low (heparan sulfate proteoglycans, HSPG) affinity. VEGF165 and VEGF121 working through VEGFR-2 stimulated nitric oxide (NO) production at low doses (0.1-1 nM), whereas only VEGF165 at high doses (10-100 nM) increased thymidine incorporation. 1 nM VEGF165 and VEGF121 induced in GENC a significant peak of inducible NO synthase (iNOS) production and, at a lower level, of endothelial NOS (eNOS). The copresence of VEGF165 with aminoguanidine (iNOS inhibitor) determined an increase of eNOS and a significant increase in thymidine incorporation. Advanced glycation end products (AGEs) working through specific receptors (RAGE) up-regulated the expression of VEGFR-2, decreased the expression of HSPG sites and reduced GENC growth. These results identify in GENC VEGFR-2 as a mediator of iNOS and eNOS release under control of VEGF, whereas HSPG binding sites seem to mediate the weak growth effect. The presence of AGEs, up-regulating the VEGFR-2 and decreasing HSPG sites might participate to the block of glomerular angiogenesis addressing the VEGF effects on glomerular permeability.     

Raw vegetable food containing high cyclo (his-pro) improved insulin sensitivity and body weight control

Cyclo (his-pro), controlled-energy diet, soy protein hydrolysate (SPH), and raw vegetable food (RVF) are known to improve insulin sensitivity and body weight (BW) control. Enhancement of high cyclo (his-pro) content in SPH (HCS) was performed by refluxing SPH with 1 N KH(2)CO(3) dissolved in 70% ethanol for 2 weeks at room temperature. Using this material, we examined the effects of HCS plus RVF on glucose metabolism and BW control in genetically diabetic Goto-Kakizaki (G-K) and insulin-resistant aged overweight Sprague-Dawley (S-D) rats. Thirty 7-week-old G-K rats and 18 16- to 18-month-old S-D rats were divided into 3 groups and treated with normal chow (NC), RVF diet, or HCS diet for 8 weeks. Raw vegetable food diet was made of 1:3 RVF and 2:3 NC; HCS diet was made of 1:27 portion HCS, 8:27 RVF, and 2:3 NC. Oral glucose tolerance significantly improved in both RVF- (P<.01) and HCS-treated (P<.001) G-K rats and worsened in NC-fed rats compared with the baseline values. Similarly, oral glucose tolerance also improved in aged overweight S-D rats when treated with RVF (P<.05) and with HCS (P<.01), compared with the baseline values. Although HCS diet treatment very significantly lowered fed plasma insulin levels compared with NC diet treatment in G-K rats (P<.01), RVF diet treatment alone did not decrease plasma insulin levels. In contrast, there was no change of insulin levels in overweight aged S-D rats after either RVF or HCS diet treatment. Postfeeding glucose levels in G-K rats fed RVF or HCS significantly fell, compared with the rats fed NC (P<.05). Interestingly, fasting blood glucose levels in RVF- or HCS-fed rats were very significantly lower than in NC-fed rats (P<.001). There was no change of blood glucose levels in S-D rats due to treatments with different diet. In G-K rats, food intake did not decrease during the first 3 weeks but fell very significantly from the fifth to eighth weeks with RVF (P<.01) and HCS (P<.001) treatments in G-K rats. However, food intake reduction in aged S-D rats was shown only for the HCS-treated rat group (P<.05). Water intake slightly decreased in G-K rats with either RVF or HCS treatment (P<.05) but very significantly decreased in S-D rats with HCS treatment (P<.01). Body weight gain in young G-K rats and BW in aged S-D rats significantly decreased only when rats were treated with HCS diet (P<.05). These data suggest that regular consumption of HCS diet helps to control blood glucose metabolism in diabetic G-K rats and BW control in aged obese S-D rats.     

Immunocytochemical localization of a neuron-specific diacylglycerol kinase beta and gamma in the developing rat brain

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA) and is, therefore, a potential terminator of DG signaling. DG and PA are important intracellular second messengers. DG directly binds protein kinase C (PKC) then activates this multifunctional enzyme. Ca2+-dependent and brain-specific DGKs, alpha, beta, and gamma, are suggested to play pivotal roles in the central nervous system. To elucidate the DGK function in neuronal development, we studied the developmental changes of DGKalpha, beta, and gamma in the postnatal rat brain. By immunoblot analysis, DGKalpha and gamma subtypes were present at birth and then gradually increased, while DGKbeta was not present at birth or postnatal day 3, then increased rapidly from day 14 to reach maximum at day 28. Immunohistochemically, DGKbeta and gamma were distributed in different brain regions. In most brain regions, DGKgamma showed sustained expression throughout the postnatal developmental periods. Interestingly, a temporal expression of DGKgamma was observed in the medial geniculate nucleus during day 3 to 14, and a delay of DGKgamma expression was seen in Purkinje cells, which was coincident with dendritic growth of Purkinje cells. In the hippocampal pyramidal cell, both DGKbeta and gamma were abundant but subcellular localization was different. DGKgamma localized in the cytosol while DGKbeta localized along the membrane structure. These findings suggest that each DGK subtype has a spatio-temporally different function in the developmental neurons.     

Human glioblastomas overexpress ADAMTS-5 that degrades brevican

Selective cleavage of the Glu³⁹⁵-Ser³⁹⁶ bond of brevican, one of the major proteoglycans in adult brain tissues, is thought to be important for glioma cell invasion. Our previous biochemical study demonstrated that ADAMTS-4, a member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, has such an activity. In the present study, we examined brevican-degrading activities of ADAMTS-1, -4 and -5 at the cellular level, and their expression and localization in human glioma tissues. In 293T transfectants expressing ADAMTS-4 or ADAMTS-5, brevican was cleaved into two major fragments in an identical pattern, but no such degradation was observed with ADAMTS-1 transfectants. When the expression levels of these ADAMTS species were examined by real-time quantitative PCR, only ADAMTS-5 was found to be overexpressed in glioblastoma tissues compared to control normal brain tissues (P <0.05). In situ hybridization and immunohistochemistry demonstrated that ADAMTS-5 is expressed predominantly in glioblastoma cells. Forced expression of ADAMTS-5 in glioma cell lines stimulated cell invasion. These results demonstrate for the first time that ADAMTS-5 is capable of degrading brevican and is overexpressed in glioblastoma cells, and suggest that ADAMTS-5 may play a role in glioma cell invasion through the cleavage of brevican.     

Effects of aging on regional cerebral blood flow assessed by using technetium Tc 99m hexamethylpropyleneamine oxime single-photon emission tomography with 3D stereotactic surface projection analysis

OBJECTIVES: Although many previous reports have described age-related changes in regional cerebral blood flow (rCBF), none has used 3D stereotactic surface projection (3D-SSP) analysis, which is able to detect subtle and significant changes in rCBF. METHODS: The subjects were 31 healthy volunteers (16 men and 15 women; 50-79 years of age) without abnormal MR imaging and MR angiographic findings, cognitive impairment, or depression. For each subject, rCBF was evaluated by using technetium Tc 99m-radiolabeled hexamethylpropyleneamine oxime single-photon emission CT. Maps of rCBF were compared among different age groups (50-59, 60-69, and 70-79 years of age) by using 3D-SSP. The mean z score for each gyrus was calculated for each age group by using a recently developed stereotactic extraction estimation method. RESULTS: Significant age-related reductions in rCBF were seen in the bilateral cingulate gyri, left inferior gyrus, bilateral medial frontal gyri, left subcallosal gyrus, and left superior temporal gyrus. Extensive and constant reduction in rCBF occurred with increasing age in the bilateral anterior cingulate gyri, and the mean z score for this region was the highest among all the regions examined. CONCLUSION: The 3D-SSP analysis revealed that the greatest reduction in rCBF occurred within the bilateral anterior cingulate gyri in normal middle-aged and older subjects.     

Extraskeletal osteosarcoma: Extensive tumor thrombus on fused PET-CT images

A 30-year-old woman developed extraskeletal osteosarcoma in the right buttock and thigh. Radiographs and unenhanced computed tomography (CT) showed a large, multilobulated mass accompanied by mineralized matrix. Contrast-enhanced CT and magnetic resonance (MR) images showed extensive tumor thrombus in the right internal- and external iliac veins. Co-registered positron emission tomography (PET) and CT images showed abnormal F-18 2-fluoro-2-deoxy-D-glucose (FDG) uptake in the tumor thrombus. PET study in our patient provided information concerning disease extent and viability of tumor thrombus.     

Binding and internalization of Clostridium perfringens iota-toxin in lipid rafts

Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). The oligomer of Ib formed in membranes induces endocytosis. We examined the binding and internalization of Ib by using Cy3-labeled Ib. Labeled Ib was retained at the membranes of MDCK cells for 60 min of incubation at 37 degrees C, and later it was detected in cytoplasmic vesicles. To determine whether Ib associates with lipid rafts, we incubated MDCK cells with Ib at 4 or 37 degrees C and fractionated the Triton-insoluble membranes. An Ib complex of 500 kDa was localized at 37 degrees C to the insoluble fractions that fulfilled the criteria of lipid rafts, but it did not form at 4 degrees C. The amount of complex in the raft fraction reached a maximum after 60 min of incubation at 37 degrees C. When the cells that were preincubated with Ib at 4 degrees C were incubated at 37 degrees C, the complex was detected in the raft fraction. The treatment of MDCK cells with methyl-beta-cyclodextrin reduced the localization of the Ib complex to the rafts and the rounding of the cells induced by Ia plus Ib. When 125I-labeled Ia was incubated with the cells in the presence of Ib at 37 degrees C, it was localized in the raft fraction. Surface plasmon resonance analysis revealed that Ia binds to the oligomer of Ib. We conclude that Ib binds to a receptor in membranes and then moves to rafts and that Ia bound to the oligomer of Ib formed in the rafts is internalized.     

Comparison between sketch map and pointing methods for evaluating spatial cognitive ability of secondary school students with mental retardation

A study was conducted to clarify the spatial ability of secondary school students with mental retardation. Two Experiments-1 and 2-were carried out to test the students' spatial knowledge using a sketch map and a pointing task. In Experiment 1, 14 students (mean IQ and SD, 57.69 and 14.13 respectively) participated and were asked to draw sketch maps of their school, and their route from school to home, and to point to landmarks displayed in photos with their finger. Only five of 13 maps of the school, and four of 14 maps of the route home were drawn without heavy distortion. However, the results for pointing out landmarks were fairly good (mean angular error, 26.86). In Experiment 2, 10 students at a different school (mean IQ and SD, 35.4 and 10.3 respectively) participated and were asked to complete the same tasks. Although the sketch maps and pointing performance were not accurate, the difference in accuracy between landmarks inside and outside the school indicated that the students had a better grasp of spatial representation when space was familiar and limited. The difference in results between the sketch map and pointing tasks implies that the two tasks require different spatial representations and cognitive processes.