Polymerogenic neuroserpin causes mitochondrial alterations and activates NFκB but not the UPR in a neuronal model of neurodegeneration FENIB.

The neurodegenerative condition FENIB (familiar encephalopathy with neuroserpin inclusion bodies) is caused by heterozygous expression of polymerogenic mutant neuroserpin (NS), with polymer deposition within the endoplasmic reticulum (ER) of neurons. We generated transgenic neural progenitor cells (NPCs) from mouse fetal cerebral cortex stably expressing either the control protein GFP or human wild type, polymerogenic G392E or truncated (delta) NS. This cellular model makes it possible to study the toxicity of polymerogenic NS in the appropriated cell type by in vitro differentiation to neurons. Our previous work showed that expression of G392E NS in differentiated NPCs induced an adaptive response through the upregulation of several genes involved in the defence against oxidative stress, and that pharmacological reduction of the antioxidant defences by drug treatments rendered G392E NS neurons more susceptible to apoptosis than control neurons. In this study, we assessed mitochondrial distribution and found a higher percentage of perinuclear localisation in G392E NS neurons, particularly in those containing polymers, a phenotype that was enhanced by glutathione chelation and rescued by antioxidant molecules. Mitochondrial membrane potential and contact sites between mitochondria and the ER were reduced in neurons expressing the G392E mutation. These alterations were associated with a pattern of ER stress that involved the ER overload response but not the unfolded protein response. Our results suggest that intracellular accumulation of NS polymers affects the interaction between the ER and mitochondria, causing mitochondrial alterations that contribute to the neuronal degeneration seen in FENIB patients.

Comparison Between Folic Acid and gH625 Peptide-Based Functionalization of Fe3O4 Magnetic Nanoparticles for Enhanced Cell Internalization.

A versatile synthetic route based on magnetic Fe3O4 nanoparticle (MNP) prefunctionalization with a phosphonic acid monolayer has been used to covalently bind the gH625 peptide on the nanoparticle surface. gH625 is a membranotropic peptide capable of easily crossing the membranes of various cells including the typical human blood-brain barrier components. A similar synthetic route was used to prepare another class of MNPs having a functional coating based on PEG, rhodamine, and folic acid, a well-known target molecule, to compare the performance of the two cell-penetrating systems (i.e., gH625 and folic acid). Our results demonstrate that the uptake of gH625-decorated MNPs in immortalized human brain microvascular endothelial cells after 24 h is more evident compared to folic acid-functionalized MNPs as evidenced by confocal laser scanning microscopy. On the other hand, both functionalized systems proved capable of being internalized in a brain tumor cell line (i.e., glioblastoma A-172). These findings indicate that the functionalization of MNPs with gH625 improves their endothelial cell internalization, suggesting a viable strategy in designing functional nanostructures capable of first crossing the BBB and, then, of reaching specific tumor brain cells.

Blood-Brain Barrier in a Haemophilus influenzae Type a In Vitro Infection: Role of Adenosine Receptors A2A and A2B.

The blood-brain barrier (BBB) is mainly made up of tightly connected microvascular endothelial cells (BMECs), surrounded by pericytes (BMPCs) which regulate BBB tightness by providing soluble factors that control endothelial proliferation. Haemophilus influenzae type a (Hia) is able to reach the BBB, crossing it, thus causing meningitis. In this study, by using an in vitro model of BBB, performed with human BMECs and human BMPCs in co-culture, we demonstrated that, after Hia infection, the number of hBMPCs decreased whereas the number of hBMECs increased in comparison with non-infected cells. SEM and TEM images showed that Hia was able to enter hBMECs and reduce TEER and VE-cadherin expression. When the cells were infected in presence of SCH58261 and PSB603 but not DPCPX, an increase in TEER values was observed thus demonstrating that A2A and A2B adenosine receptors play a key role in BBB dysfunction. These results were confirmed by the use of adenosine receptor agonists CGS21680, CCPA, and NECA. In infected co-cultures cAMP and VEGF increased and TEER reduction was counter-balanced by VEGF-R1 or VEGF-R2 antibodies. Moreover, the phosphorylated CREB and Rho-A significantly increased in infected hBMECs and hBMPCs and the presence of SCH58261 and PSB603 significantly abrogated the phosphorylation. In conclusion, this study demonstrated that the infection stimulated A2A and A2B adenosine receptors in hBMECs and hBMPCs thus inducing the pericytes to release large amounts of VEGF. The latter could be responsible for both, pericyte detachment and endothelial cell proliferation, thus provoking BBB impairment.

Ferritin-supported lipid bilayers for triggering the endothelial cell response.

Hybrid nanoassemblies of ferritin and silica-supported lipid bilayers (ferritin-SLBs) have been prepared and tested for the adhesion, spreading and proliferation of retinal microvascular endothelial cells (ECs). Lipid membranes with varying surface charge were obtained by mixing cationic 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC) with zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at increasing POPC/POEPC ratios. The supported bilayer formation and their subsequent interaction processes with ferritin were studied at the pH of 7.4 at different protein concentrations, by using the quartz crystal microbalance with dissipation monitoring and by atomic force microscopy. Both kinetics and viscoelastic parameters of the protein-lipid membrane interface were scrutinized, as well as surface coverage. Phase-contrast optical microscopy analyses of the ferritin-SLBs substrates after their interaction with endothelial cells evidenced the highest cell adhesion (2-4h of incubation time) and proliferation (from 24h to 5 days) for the membranes of POPC/POEPC (75:25 ratio). Moreover, ferritin increased both cell adhesion and proliferation in comparison to control glass (respectively 1.5- and 1.75-fold) as well as proliferation in comparison to bare POPC/POEPC (95:5 ratio) (2 fold). Results are very promising in the goal of modulating the endothelial cell response through the interplay of viscoelastic/charge properties of the solid-supported membranes and the SLB-conditioned ferritin activity.

Role of cytosolic and calcium independent phospholipases A(2) in insulin secretion impairment of INS-1E cells infected by S. aureus.

Cytosolic PLA2 (cPLA2) and Ca(2+)-independent PLA2 (iPLA2) play a significant role in insulin ß-cells secretion. Bacterial infections may be responsible of the onset of diabetes. The mechanism by which Staphylococcus aureus infection of INS-1 cells alters glucose-induced insulin secretion has been examined. After acute infection, insulin secretion and PLA2 activities significantly increased. Moreover, increased expressions of phospho-cPLA2, phospho-PKCα and phospho-ERK 1/2 were observed. Chronic infection causes a decrease in insulin release and a significant increase of iPLA2 and COX-2 protein expression. Moreover, insulin secretion in infected cells could be restored using specific siRNAs against iPLA2 isoform and specific COX-2 inhibitor.

Asthenozoospermia and membrane remodeling enzymes: a new role for phospholipase A2.

Phosholipase A2 (PLA2 ) activity in the seminal plasma and in sperm heads is closely related to sperm motility and male fertility. Therefore, the purpose of this study was to investigate the possible involvement of different isoforms of phospholipase in asthenozoospermia. To accomplish this, cPLA2 , phospho-cPLA2 , iPLA2 , and sPLA2 were evaluated by immunofluorescence and immunoblot analyses in spermatozoa obtained from 22 normozoospermic men and 28 asthenozoospermic patients. We found significant differences in cPLA2 and its phosphorylated/activated form, iPLA2 , and sPLA2 content and distribution in normal and asthenozoospermic patients. cPLA2 was localized in heads, midpieces, and tails of all spermatozoa as constitutive enzyme, less expressed in the tail of spermatozoa with low progressive motility. While active phospho-cPLA2 distribution was homogeneous throughout the cell body of control-donor spermatozoa, lower levels were detected in the tails of asthenozoospermic patients, as opposed to its strong presence in heads. Low immunofluorescence signal for iPLA2 was found in astenozoospermic patients, whereas sPLA2 was significantly lower in the heads of asthenozoospermic patients. Spermatozoa with low progressive motility showed differences both in terms of total specific activity and of intracellular distribution. cPLA2 , iPLA2 , and sPLA2 specific activities correlated positively and in a significantly manner with sperm progressive motility both in normozoospermic men and asthenozoospermic patients. In conclusion, PLA2 s are expressed in different areas of human spermatozoa. Spermatozoa with low motility showed differences in total specific activity and enzyme distributions. We speculated that PLA2 expression and/or different distribution could be potential biomarkers of asthenozoospermia, one of the major causes of male factor infertility.

Klebsiella pneumoniae induces an inflammatory response in an in vitro model of blood-retinal barrier.

Klebsiella pneumoniae has become an important pathogen in recent years. Although most cases of K. pneumoniae endogenous endophthalmitis occur via hematogenous spread, it is not yet clear which microbial and host factors are responsible for the ability of K. pneumoniae to cross the blood-retinal barrier (BRB). In the present study, we show that in an in vitro model of BRB based on coculturing primary bovine retinal endothelial cells (BREC) and primary bovine retinal pericytes (BRPC), K. pneumoniae infection determines changes of transendothelial electrical resistance (TEER) and permeability to sodium fluorescein. In the coculture model, bacteria are able to stimulate the enzyme activities of endothelial cytosolic and Ca(2+)-independent phospholipase A2s (cPLA2 and iPLA2). These results were confirmed by the incremental expression of cPLA2, iPLA2, cyclo-oxygenase-1 (COX1), and COX2 in BREC, as well as by cPLA2 phosphorylation. In supernatants of K. pneumoniae-stimulated cocultures, increases in prostaglandin E2 (PGE2), interleukin-6 (IL-6), IL-8, and vascular endothelial growth factor (VEGF) production were found. Incubation with K. pneumoniae in the presence of arachidonoyl trifluoromethyl ketone (AACOCF3) or bromoenol lactone (BEL) caused decreased PGE2 and VEGF release. Scanning electron microscopy and transmission electron microscopy images of BREC and BRPC showed adhesion of K. pneumoniae to the cells, but no invasion occurred. K. pneumoniae infection also produced reductions in pericyte numbers; transfection of BREC cocultured with BRPC and of human retinal endothelial cells (HREC) cocultured with human retinal pericytes (HRPC) with small interfering RNAs (siRNAs) targeted to cPLA2 and iPLA2 restored the pericyte numbers and the TEER and permeability values. Our results show the proinflammatory effect of K. pneumoniae on BREC, suggest a possible mechanism by which BREC and BRPC react to the K. pneumoniae infection, and may provide physicians and patients with new ways of fighting blinding diseases.

Late presentation of HIV infection: predictors of delayed diagnosis and survival in Eastern Sicily.

OBJECTIVES: Across Europe, more than one third of patients are diagnosed with HIV infection late. Late presentation for care has been associated with higher risk of clinical progression and mortality. In the present study, we evaluated the prevalence, epidemiological characteristics and survival probability of patients with late and very late presentation, newly diagnosed with HIV infection in Catania, Italy, from 1985 to 2010. PATIENTS AND METHODS: According to the European Consensus definition, Late Presenters (LP) were defined as subjects presenting for care with a CD4+ T-cell count below 350 cells/µl or with an AIDS-defining event, regardless of CD4+ T-cell count; patients with advanced HIV disease (Very Late Presenters) (VLP) were those presenting with a CD4+ T-cell count below 200 cells/µl or with an AIDS-defining event, regardless of CD4+ T-cell count. RESULTS: 620 patients were included in the study. 345 (55.6%) subjects were LP, 35% of them were asymptomatic; 246 (39.7%) were VLP. In univariate analysis, late presentation was related to age (p < 0.001), to heterosexual exposure to HIV infection (70% of heterosexual subjects were LP) (p < 0.005) and to being diagnosed during the calendar period from 1991 to 2000 (p < 0.001). Very late presentation was related to age (p < 0.001), male sex (p < 0.01), heterosexual risk (p < 0.001) and to being diagnosed during the calendar period from 1991 to 2000 (p < 0.001). In multivariate analysis, age (p < 0.0001), being older than 50 years old (p = 0.02), years of diagnosis 1991-1995 (p < 0.005) and 1996-2000 (p < 0.05) in the subgroup of late presenters and age (p < 0.0001), being older than 50 years old (p < 0.005), male sex (p < 0.0001), years of diagnosis 1991-1995 (p < 0.05) and 1996-2000 (p < 0.005) in the subgroup of very late presenters maintained statistical significance. The survival probability within LP and VLP group was statistically lower than no LP/VLP (log rank test p < 0.0005 and p < 0.0001, respectively). For both LP (p < 0.002) and VLP (p < 0.0001), survival probability was significantly lower in the pre-HAART era, in comparison with the period of mono/dual therapy and the HAART era. CONCLUSIONS: More than fifty percent of patients in our setting were diagnosed late with HIV infection and, consequently, treated late. Late and very late presentation were associated with lower survival probability. The implementation of strategies focused on targeted prevention efforts and HIV testing programs appears fundamental to diagnose and treat HIV infection as early as possible.

Management of temporomandibular joint ankylosis: a case report of joint replacement with piezoelectric surgery.

AIM: Temporomandibular joint (TMJ) ankylosis is a joint disorder which refers to bone or fibrous adhesion of the anatomic joint components and the ensuing loss of function. This report describes the management of a case of bilateral TMJ ankylosis in a 20-year-old patient with prosthetic replacement with the aid of a piezoelectric instrument (MECTRON (R). METHODS: The right ankylotic mass was surgically removed and replaced by a custom-made prosthesis based on data obtained from three-dimensional computed tomography (CT) reconstruction of the skull of the patient using a stereolithography model. RESULTS: At six months postoperative, the opening of the mouth was stable at 36 mm and imaging studies (CT and magnetic resonance imaging [MRI]) showed a great condylar prosthesis and surrounding tissues in addition to partial remodeling of the left TMJ. CONCLUSION: In this patient, despite the bilateral ankylosis, it was sufficient to intervene only on the right TMJ, which presented a serious bone block, with mobilization since surgery gradually restored the anatomical and functional conditions of the left TMJ compatible with normal activities of mastication and speech.

Protein kinase C activation affects, via the mRNA-binding Hu-antigen R/ELAV protein, vascular endothelial growth factor expression in a pericytic/endothelial coculture model.

PURPOSE: To explore whether, following direct contact, there is mutual influence between pericytes (PC) and endothelial cells (EC), and to establish whether protein kinase C (PKC) activation, a condition associated with hyperglycemia, can affect, via the mRNA-binding Hu-antigen R (HuR)/ELAV protein, the expression of vascular endothelial growth factor (VEGF). METHODS: PC and EC were cultured separately or in direct contact (1:1 ratio), and exposed or not to phorbol esters, a PKC activator (100 nM for 15 min). Barrier integrity was evaluated by measuring endothelial electrical resistance and permeability to sodium fluorescein. Immunocytochemistry was performed to visualize EC and PC in coculture, and to evaluate phorbol 12-myristate-13-acetate (PMA)-induced HuR translocation. PKCßI/ßII, HuR, and VEGF protein content was measured with western blotting, VEGF secretion in cell culture medium was evaluated with enzyme-linked immunosorbent assay (ELISA), and quantification of VEGF mRNA was performed with real-time quantitative PCR. RESULTS: In monocultures, VEGF mRNA/protein basal levels were more elevated in PC than in EC. However, the basal expression of VEGF protein, but not mRNA, in PC and EC was affected by culture conditions. In fact, physical contact with PC upregulated VEGF protein levels in the EC, while VEGF was downregulated in PC cocultured with EC. In this last condition, PKCßII and HuR protein basal levels were also decreased in monocultured PC. Moreover, in basal conditions, the amount of VEGF released from the coculture was higher than from the monocultures. Direct activation of PKCß induced HuR translocation from the nuclear area to the cytoplasm, and increased the protein levels of the kinase itself, HuR, and VEGF in PC and EC in both culture conditions. Concerning VEGF mRNA, PKC activation induced an increase in PKC levels only in monocultured EC and, conversely, a significant decrease in the same transcript amount in cocultured PC. PMA stimulus also led to a significant increase in VEGF secretion in coculture. CONCLUSIONS: When cocultured with PC, EC form a significantly tighter barrier than the endothelial monolayer. The physical contact leads to opposite changes in VEGF protein levels in PC and EC. In particular, in basal conditions, cocultured PC seemed to downregulate their own expression of this proproliferating factor, as well as that of PKCßII and HuR, likely to maintain the 1:1 ratio with the cohabiting EC. In mono- and cocultured PC/EC, PKC direct activation led to a similar increase in PKCßI/ßII, HuR, and VEGF protein levels, changes that may also occur at early stages of diabetic retinopathy. The release of VEGF in the medium was favored by physical contact between PC and EC and was further increased by PMA exposure. In contrast with the effects on VEGF protein, PKCß activation induced modifications in VEGF mRNA content that are different in function of the cell type and the culture conditions. These findings suggest that the changes in the VEGF protein and transcript observed in PC/EC can be ascribed to distinct and concomitant pathways. Further studies on this in vitro coculture model would be useful to better understand the PC/EC interaction in physiologic and pathological conditions.

The piezoelectric osteotomy technique compared to the one with rotary instruments in the surgery of included third molars. A clinical study.

AIM: The purpose of this study is to compare surgical times, the saving of bonytissues, the postoperative course of the extraction of included third molars in patients treated with piezoelectric surgery compared to those treated with conventional rotary instruments. METHODS: The study included 140 patients with upper and lower third molars included. 70 of them were treated with osteotomy using conventional rotary instruments (Group 1) and 70 more with osteotomy through piezosurgery Mectron® (GROUP 2). The treatment protocol was the same for both groups. The time of surgery and the saving of bonytissues in the intra-operative have been estimated; moreover, 24-48-72 hours and 7 days after the surgery, two other parameters have been estimated: facial swelling and trismus. The swelling, trismus and the saving of bony tissues were assessed by a surgery gauge. RESULTS: The average surgical time was 15 minutes in Group 1 and 20 minutes in Group 2. The savings of bonytissues was greater in Group 2 than in group 1 by 2.7 mm. The average facial swelling was 6.23 mm for group 1 and 2.86 mm for group 2 24 hours after the surgery, of 5.22 mm for group 1 and 1.76 mm for group 2 48 hours after the intervention, of 3.75 mm for Group 1 and 0.85 for group 2 72 hours after the surgery, of 0.86 mm for group 1 and 0.12 for group 2. The average trismus was of 14.76 mm in Group 1 and 11.15 mm in Group 2. Statistical analysis showed a significant reduction of swelling, trismus and a bigger saving of bony tissues in Group 2 and a reduction in surgical time in Group 1. CONCLUSION: The osteotomy technique with a piezoelectric instrument has produced a significant reduction in facial swelling, trismus and a bigger saving of bony tissues than with conventional rotary instruments. A slight lengthening in terms of surgical time has been recorded, compared to the use of conventional rotary instruments.

Benefits of piezoelectric surgery in oral and maxillofacial surgery. Review of literature.

Piezoelectric surgery is based on the use of ultrasound for the cutting of bones. It represents an innovative technique, as it offers the maxillofacial surgeon the opportunity of making precise bone cuts without damaging any soft tissue, minimizing the invasiveness of surgical procedure, and the opportunity of working in a field which is almost totally blood-free. It reduces the impact on soft tissues (vessels and nerves) which lie adjacent to the areas of treatment. Compared to traditional methods, it enables optimal healing because it reduces the postsurgery swelling and discomfort. In this article the authors realized a review of the literature.

Oral vinorelbine as first line chemotherapy in unfit elderly patients with hormone-refractory prostate cancer.

Hormone-refractory prostate cancer (HRPC) is a rapidly progressive disease which produces considerable morbidity and involves mostly men over 70, often comorbid and with poor tolerance to chemotherapy. Low-toxicity chemotherapy is a reasonable option in this setting. Vinorelbine and a corticosteroid show activity and clinical benefit responses in HRPC. An oral regimen is preferable for elderly patients. This study aimed to evaluate safety, prostate-specific antigen (PSA) response, clinical benefit and progression-free survival in chemonaive elderly HRPC patients. 33 men, median age 78.2, were treated with oral vinorelbine 60 mg/m2 days 1 and 8 every 3 weeks, escalable to 80 mg/m2 after the first cycle, and prednisone 5 mg b.i.d. The main toxicity was hematopoietic (mild at 60 mg/m2 and moderate at 80 mg/m2). Of 27 evaluable patients, 9 (33%) had PSA responses and 9 had clinical benefit, PSA-correlated in 5 cases (56%). Median progression-free survival was 13.4 weeks, median overall survival 45 weeks. Oral vinorelbine plus prednisone is safe and has moderate activity, with biochemical and clinical responses in about one-third of patients and could be an option in unfit elderly HRPC patients.

Loss of aromatase cytochrome P450 function as a risk factor for Parkinson's disease?

The final step in the physiological synthesis of 17beta estradiol (E(2)) is aromatization of precursor testosterone by a CYP19 gene product, cytochrome P450 estrogen aromatase in the C19 steroid metabolic pathway. Within the central nervous system (CNS) the presence, distribution, and activity of aromatase have been well characterized. Developmental stage and injury are known modulators of brain enzyme activity, where both neurons and glial cells reportedly have the capability to synthesize this key estrogenic enzyme. The gonadal steroid E(2) is a critical survival, neurotrophic and neuroprotective factor for dopaminergic neurons of the substantia nigra pars compacta (SNpc), the cells that degenerate in Parkinson's disease (PD). In previous studies we underlined a crucial role for the estrogenic status at the time of injury in dictating vulnerability to the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our ongoing studies address the contribution of brain aromatase and extragonadal E(2) as vulnerability factors for PD pathology in female brain, by exposing aromatase knockout (ArKO, -/-) female mice which are unable to synthesize estrogens to MPTP. Our initial results indicate that aromatase deficiency from early embryonic life significantly impairs the functional integrity of SNpc tyrosine hydroxylase-positive neurons and dopamine transporter innervation of the caudate-putamen in adulthood. In addition, ArKO females exhibited a far greater vulnerability to MPTP-induced nigrostriatal damage as compared to their Wt type gonadally intact and gonadectomized counterparts. Characterization of this novel implication of P450 aromatase as determining factor for PD vulnerability may unravel new avenues for the understanding and development of novel therapeutic approaches for Parkinson's disease.

High glucose and advanced glycation end products induce phospholipid hydrolysis and phospholipid enzyme inhibition in bovine retinal pericytes.

In the present study, we investigated the possible role of oxidative stress and the modulation of phospholipid turnover in two related models of pericyte injury, i.e., treatment with high glucose or advanced glycation end products (AGEs). Growing microcapillary pericytes from bovine retinas in culture were incubated, for 3 weeks, with 20-50 mM glucose or 2-20 microM AGEs, and peroxidation parameters (malondialdehyde, conjugated diene, hydroperoxide, glutathione (GSH) levels and lactate dehydrogenase (LDH) release) were evaluated. Arachidonate (AA) and choline release from membrane phospholipids was determined in pericytes prelabeled with [1-(14)C]arachidonate and [Me-(3)H]choline, respectively, and stimulated with elevated glucose or AGEs for 30 min or 2 h. [1-(14)C]arachidonate and [Me-(3)H]choline incorporation into phospholipids, for 2 h and 3 h respectively, was also studied in conditioned and serum-starved cultures. Finally, lysates of treated and control cells were assayed for cytosolic phospholipase A(2) (cPLA(2)), acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (AT), CTP:phosphocholine cytidylyltransferase (CT) and microsomal choline phosphotransferase (CPT) enzyme activities. We found that high glucose and AGEs caused neither significant production of reactive oxygen species nor cell toxicity or death, unlike other cell types. Both agents had no significant effect on the cellular ultrastructure, evaluated by light and electron microscopy, AA incorporation and release, cytosolic phospholipase A(2) (cPLA(2)) and AT activities. On the contrary, choline incorporation into phosphatidylcholine, CT and CPT activities were significantly reduced either by 50 mM glucose or 20 microM AGEs. Simultaneously, [Me-(3)H]choline release was significantly stimulated by both agents. We conclude that prolonged treatments with high glucose or AGEs are not able to induce oxidative injury in bovine retinal capillary pericytes. Nevertheless, they do induce phospholipid hydrolysis and phospholipid enzyme activity inhibition.

Amyloid beta(1-42) and its beta(25-35) fragment induce in vitro phosphatidylcholine hydrolysis in bovine retina capillary pericytes.

We describe the inhibitory effect of full-length Abeta(1-42) and Abeta(25-35) fragment of amyloid-beta peptide on phosphatidylcholine (PtdCho) metabolism in bovine retina capillary pericytes. Cell cultures were incubated with Abetas for 24 h. Peroxidation indices (malondialdehyde and lactate dehydrogenase release) significantly increased after 20-50 microM Abeta(1-42) or Abeta(25-35) treatment. In addition, [Me-3H]choline incorporation into PtdCho strongly decreased while either 3H-choline or 14C-arachidonic acid release from prelabeled cells increased, indicating PtdCho hydrolysis. The effect was very likely due to prooxidant action of both Abeta peptides. Reversed-sequence Abeta(35-25) peptide did not depress 3H-choline incorporation nor stimulate PtdCho breakdown. With addition of Abetas at low concentrations (2-20 microM) to pericytes, marked ultrastructural changes, well connected to metabolic alterations, emerged including shrinkage of cell bodies, retraction of processes, disruption of the intracellular actin network. Cells treated with higher concentrations (50-200 microM) displayed characteristics of necrotic cell death. The data suggest that: (a) Abeta(1-42) and Abeta(25-35) peptides may modulate phospholipid turnover in microvessel pericytes; (b) together with endothelial cells, pericytes could be the target of vascular damage during processes involving amyloid accumulation.

t-Butyl hydroperoxide and oxidized low density lipoprotein enhance phospholipid hydrolysis in lipopolysaccharide-stimulated retinal pericytes.

Free radicals induced by organic peroxides or oxidized low density lipoprotein (oxLDL) play a critical role in the development of atherosclerosis. In investigating this process, and the concomitant inflammatory response, the role of pericytes, cells supporting the endothelial ones in blood vessels, has received little attention. In this study we tested the hypothesis that tert-butyl hydroperoxide (t-BuOOH) and oxLDL, administered in sublethal doses to the culture medium of retinal pericytes, function as prooxidant signals to increase the stimulation of the peroxidation process induced by lipopolysaccharide (LPS). Confluent cell monolayers were exposed to t-BuOOH (25-400 microM), native LDL or oxLDL (3.4-340 nmol hydroperoxides/mg protein, 1-100 micro). LPS (1 microg/ml), t-BuOOH (200 microM), and oxLDL (100 microM), but not native LDL, incubated for 24 h with cells, markedly increased lipid peroxidation, cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release in a time- and dose-dependent manner. AACOCF(3), a potent cPLA2 inhibitor, and the antioxidant alpha-tocopherol strongly inhibited the prooxidant-stimulated AA release. Long-term exposure to maximal concentrations of t-BuOOH (400 microM) or oxLDL (100 microM) had a sharp cytotoxic effect on the cells, described by morphological and biochemical indices. The presence of t-BuOOH or oxLDL at the same time, synergistically increased phospholipid hydrolysis induced by LPS alone. 400 microM t-BuOOH or 100 microM oxLDL had no significant effect on the stimulation of an apoptosis process estimated by DNA laddering and light and electron microscopy. The results indicate that (i) pericytes may be the target of extensive oxidative damage; (ii) activation of cPLA2 mediates AA liberation; (iii) as long-term regulatory signals, organic peroxide and specific constituents of oxLDL increase the pericyte ability to degrade membrane phospholipids mediated by LPS which was used, in the present study, to simulate in vitro an inflammatory burst in the retinal capillaries.

Expression of the Xvax2 gene demarcates presumptive ventral telencephalon and specific visual structures in Xenopus laevis.

vax2 is a recently isolated homeobox gene, that plays an important role in controlling the dorso-ventral patterning of the retina. In this paper we present a thorough description of the Xvax2 expression pattern all along Xenopus embryogenesis, and compare this pattern in detail to that shown by Xvax1b and Xpax2, two genes also involved in ventral eye development. At early neurula stages, while Xpax2 starts to be expressed within the eye field, both Xvax2 and Xvax1b are exclusively activated in the presumptive ventral telencephalon. Since midneurula stages, Xvax2 and Xvax1b are also transcribed in the medial aspect of the eye field. At tailbud and tadpole stages, Xvax2, Xvax1b and Xpax2 expression overlaps in the optic stalk and nerve and in the optic disk, while Xvax2 and Xvax1b also display specific activation domains in the ventral retina as well as in the ventral telencephalon and diencephalon. Finally, during metamorphosis a high level of both Xvax2 and Xvax1b transcription is maintained in the optic chiasm. In addition, Xvax1b is transcribed in the ventral hypothalamus and in the hypophysis, whereas a strong Xvax2 expression is retained in the ventral portion of the mature retina.

Homeobox genes in the genetic control of eye development.

Vertebrate eye formation is a complex process which involves early specification of the prospective eye territory, induction events, patterning along the polarity axes and regional specification, to bring about the proper morphogenetic movements, cell proliferation, cell differentiation and neural connections allowing visual function. The molecular machinery underlying such complex developmental events is presently under an intense research scrutiny and many associated genetic factors have been isolated and characterized. These studies produced striking knowledge in the field, especially with respect to uncovering the role of key genes and their possible evolutionary conservation. Presently, a major task is to define the complex interactions connecting the multiplicity of molecular players that regulate eye development. We recently identified two homeobox genes, Xrx1 and Xvax2, and studied their function by using the Xenopus embryo as a developmental model system. Xrx1 and Xvax2 control key aspects of eye development. In particular, Xrx1 appears to play a role in the early specification of anterior neural regions fated to give rise to retina and forebrain structures, and in promoting cell proliferation within these territories. On the other hand, Xvax2 is involved in regulating the eye proximo-distal and/or dorsoventral polarity, and the morphogenetic movements taking place during formation of the optic stalk and cup. Here we review the experimental results addressing the roles of Xrx1 and Xvax2 and their vertebrate orthologues, and discuss their relationship with other molecules also playing a related function in eye development.

Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis.

We describe the cloning, expression pattern and functional overexpression analysis of Xotx5b, a new member of the Otx gene family in Xenopus laevis. Early expression of Xotx5b resembles that of Xotx2, being detected in the organizer region at early gastrula stage, and, shortly after, also in anterior neuroectoderm. During neurula stages Xotx5b exhibits a changing and dynamic pattern of expression. After neural tube closure, Xotx5b is expressed in the eye and pineal gland, both involved in photoreception. Overexpression of Xotx5b has a similar effect to that of Xotx2, producing posterior truncations and inducing ectopic cement gland and neural tissue in whole embryos. In animal cap assays, Xotx5b and Xotx2 are both able to activate XAG, to strongly suppress the expression of the epidermal marker XK81, and to reciprocally activate each other. Finally, in einsteck transplantation assays, Xotx5b is able to respecify a tail/trunk organizer to a head organizer.