Human munc13 Is a Diacylglycerol Receptor that Induces Apoptosis and May Contribute to Renal Cell Injury in Hyperglycemia

We have previously shown that human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, we have investigated the subcellular localization of hmunc13 in a transiently transfected renal cell line, opossum kidney cells. We have found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant in which the diacylglycerol (C1) binding domain is absent exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. We conclude that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. We have also demonstrated that munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, our data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia.

Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells.

Orally administered antigens induce a state of immunologic hyporesponsiveness termed oral tolerance. Different mechanisms are involved in mediating oral tolerance depending on the dose fed. Low doses of antigen generate cytokine-secreting regulatory cells, whereas high doses induce anergy or deletion. We used mice transgenic for a T-cell receptor (TCR) derived from an encephalitogenic T-cell clone specific for the acetylated N-terminal peptide of myelin basic protein (MBP) Ac-1-11 plus I-Au to test whether a regulatory T cell could be generated from the same precursor cell as that of an encephalitogenic Th1 cell and whether the induction was dose dependent. The MBP TCR transgenic mice primarily have T cells of a precursor phenotype that produce interleukin 2 (IL-2) with little interferon gamma (IFN-gamma), IL-4, or transforming growth factor beta (TGF-beta). We fed transgenic animals a low-dose (1 mg x 5) or high-dose (25 mg x 1) regimen of mouse MBP and without further immunization spleen cells were tested for cytokine production. Low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-beta, whereas minimal secretion of these cytokines was observed with high-dose feeding. Little or no change was seen in proliferation or IL-2/IFN-gamma secretion in fed animals irrespective of the dose. To demonstrate in vivo functional activity of the cytokine-secreting cells generated by oral antigen, spleen cells from low-dose-fed animals were adoptively transferred into naive (PLJ x SJL)F1 mice that were then immunized for the development of experimental autoimmune encephalomyelitis (EAE). Marked suppression of EAE was observed when T cells were transferred from MBP-fed transgenic animals but not from animals that were not fed. In contrast to oral tolerization, s.c. immunization of transgenic animals with MBP in complete Freund's adjuvant induced IFN-gamma-secreting Th1 cells in vitro and experimental encephalomyelitis in vivo. Despite the large number of cells reactive to MBP in the transgenic animals, EAE was also suppressed by low-dose feeding of MBP prior to immunization. These results demonstrate that MBP-specific T cells can differentiate in vivo into encephalitogenic or regulatory T cells depending upon the context by which they are exposed to antigen.

Tauroursodeoxycholic acid prevents retinal degeneration in transgenic P23H rats

Purpose. To evaluate the preventive effect of tauroursodeoxycholic acid (TUDCA) on photoreceptor degeneration, synaptic connectivity and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). Methods. P23H line-3 rats were injected with TUDCA once a week from postnatal day (P)21 to P120, in parallel with vehicle-administered controls. At P120, functional activity of the retina was evaluated by electroretinographic (ERG) recording. The effects of TUDCA on the number, morphology, integrity, and synaptic connectivity of retinal cells were characterized by immunofluorescence confocal microscopy. Results. The amplitude of ERG a- and b-waves was significantly higher in TUDCA-treated animals under both scotopic and photopic conditions than in control animals. In the central area of the retina, TUDCA-treated P23H rats showed threefold more photoreceptors than control animals. The number of TUNEL-positive cells was significantly smaller in TUDCA-treated rats, in which photoreceptor morphology was preserved. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in TUDCA-treated P23H rats. Furthermore, in TUDCA-treated rat retinas, the number of both rod bipolar and horizontal cell bodies, as well as the density of their synaptic terminals in the outer plexiform layer, was greater than in control rats. Conclusions. TUDCA treatment was capable of preserving cone and rod structure and function, together with their contacts with their postsynaptic neurons. The neuroprotective effects of TUDCA make this compound potentially useful for delaying retinal degeneration in RP.

Proinsulin slows retinal degeneration and vision loss in the P23H rat model of retinitis pigmentosa

Proinsulin has been characterized as a neuroprotective molecule. In this work we assess the therapeutic potential of proinsulin on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). P23H homozygous rats received an intramuscular injection of an adeno-associated viral vector serotype 1 (AAV1) expressing human proinsulin (hPi+) or AAV1-null vector (hPi−) at P20. Levels of hPi in serum were determined by enzyme-linked immunosorbent assay (ELISA), and visual function was evaluated by electroretinographic (ERG) recording at P30, P60, P90, and P120. Preservation of retinal structure was assessed by immunohistochemistry at P120. Human proinsulin was detected in serum from rats injected with hPi+ at all times tested, with average hPi levels ranging from 1.1 nM (P30) to 1.4 nM (P120). ERG recordings showed an amelioration of vision loss in hPi+ animals. The scotopic b-waves were significantly higher in hPi+ animals than in control rats at P90 and P120. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. hPi+ animals had 48.7% more photoreceptors than control animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in hPi+ P23H rats. Furthermore, in hPi+ rat retinas the number of rod bipolar cell bodies was greater than in control rats. Our data demonstrate that hPi expression preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in the P23H rat. These data strongly support the further development of proinsulin-based therapy to counteract retinitis pigmentosa.

Neuroprotective effects of the cannabinoid agonist HU210 on retinal degeneration

Cannabinoids have been demonstrated to exert neuroprotective effects on different types of neuronal insults. Here we have addressed the therapeutic potential of the synthetic cannabinoid HU210 on photoreceptor degeneration, synaptic connectivity and functional activity of the retina in the transgenic P23H rat, an animal model for autosomal dominant retinitis pigmentosa (RP). In P23H rats administered with HU210 (100 μg/kg, i.p.) from P24 to P90, ERG recordings showed an amelioration of vision loss, as compared to vehicle-administered animals. Under scotopic conditions, the maximum a-wave amplitudes recorded at P60 and P90 were higher in HU210-treated animals, as compared to the values obtained in untreated animals. The scotopic b-waves were significantly higher in treated animals than in untreated rats at P30, P60 and P90. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. HU210-treated animals had 40% more photoreceptors than untreated animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were also preserved in HU210-treated P23H rats. These results indicate that HU210 preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in P23H rats. These data suggest that cannabinoids are potentially useful to delay retinal degeneration in RP patients.

Herpesvirus-specific CD8 T cell immunity in old age: Cytomegalovirus impairs the response to a coresident EBV infection

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.

Similar antibody concentrations in Filipino infants at age 9 months, after 1 or 3 doses of an adjuvanted, 11-valent pneumoccoccal diphtheria/tetanus-Conjugated Vaccine: a randomized controlled trial

In Filipino infants, 1 dose of an adjuvanted, 11-valent pneumococcal conjugate vaccine (serotypes 1, 4, 5, 7F, 9V, 19F, and 23F conjugated to tetanus protein; and serotypes 3, 6B, 14, and 18C conjugated to diphtheria toxoid) administered alone at age 18 weeks (11PncTD1) elicited similar antibody concentrations at age 9 months as those elicited by 3 doses (11PncTD3) administered concomitantly with national program vaccines, at ages 6, 10, and 14 weeks. Geometric mean antibody concentrations ranged from 0.36 mug/mL ( for serotype 18C) to 5.81 mug/mL (for serotype 4), for the 11PncTD1 vaccine, and from 0.32 mug/mL (for serotype 18C) to 5.01 mug/mL (for serotype 19F), for the 11PncTD3 vaccine. The proportion of infants with threshold antibody concentrations greater than or equal to0.35 mug/mL was also similar (ranges, 55.6%-100% for the 11PncTD1 vaccine and 42.9%-100% for the 11PncTD3 vaccine). The functional activity of antibodies expressed as opsonophagocytic activity titers was similar in the 11PncTD1 and 11PncTD3 groups. This finding is an important one for countries with financial constraints and high pneumococcal disease burden.

Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis

The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted-cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild-type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell-surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7-fold for hP97C and 10-fold for hF101C and increased 12-fold for hY98C. [H-3]Nisoxetine binding affinities were decreased 13-fold for hP97C and 5-fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mM MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.

Characterization of two polymorphisms in the leukotriene C4 synthase gene in an Australian population of subjects with mild, moderate, and severe asthma

Background: The cysteinyl-leukotrienes (cys-LTs) are proinflammatory mediators that are important in the pathophysiology of asthma. LTC4 synthase is a key enzyme in the cys-LT biosynthetic pathway, and studies in small populations have suggested that a promoter polymorphism (A(-444)C) in the gene might be associated with asthma severity and aspirin intolerance. Objective: We sought to screen the LTC4 synthase gene for polymorphisms and to determine whether there is an association between these polymorphisms and asthma severity or aspirin sensitivity in a large, well-phenotyped population and to determine whether this polymorphism is functionally relevant. Methods: The coding regions of the LTC4 synthase gene were screened for polymorphisms and the A(-444)C polymorphism was analyzed in a large Australian white adult population of mild (n = 282), moderate (n = 236), and severe asthmatic subjects (n = 86) and nonasthmatic subjects (n = 458), as well as in aspirin-intolerant asthmatic subjects (n = 67). The functional activity of the promoter polymorphism was investigated by transient transfection of HL-60 cells with a promoter construct. Results: A new polymorphism was identified in intron 1 of the gene (IVS1-10c>a) but was not associated with asthma. Association studies showed that the A(-444)C polymorphism was weakly associated with asthma per se, but there was no association between the C-444 allele and chronic asthma severity or aspirin intolerance. A meta-analysis of all the genetic studies conducted to date found significant between-study heterogeneity in C-444 allele frequencies within different clinical subgroups. In vitro functional studies showed no significant differences in transcription efficiency between constructs containing the A(-444) allele or the C-444 allele. Conclusions: Our data confirm that, independent of transcriptional activity, the C-444 allele in the LTC4 synthase gene is weakly associated with the asthma phenotype, but it is not related to disease severity or aspirin intolerance.

Development of an in-vitro model system to investigate the mechanism of muscle protein catabolism induced by proteolysis-inducing factor

The mechanism of muscle protein catabolism induced by proteolysis-inducing factor, produced by cachexia-inducing murine and human tumours has been studied in vitro using C2C12 myoblasts and myotubes. In both myoblasts and myotubes protein degradation was enhanced by proteolysis-inducing factor after 24 h incubation. In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor. Protein degradation induced by proteolysis-inducing factor was completely attenuated in the presence of cycloheximide (1 μM), suggesting a requirement for new protein synthesis. In both myoblasts and myotubes protein degradation was accompanied by an increased expression of the α-type subunits of the 20S proteasome as well as functional activity of the proteasome, as determined by the 'chymotrypsin-like' enzyme activity. There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E214k), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor. These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia. © 2002 Cancer Research UK.

Expression of the ubiquitin-proteasome pathway and muscle loss in experimental cancer cachexia

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E2(14k), has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E2(14k) were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E2(14k) mRNA and protein levels in gastrocnemius muscle with increases of 6-8-fold for C2 and two-fold for E2(14k) between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25-27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18-20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E2(14k) mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss. © 2005 Cancer Research.

Accuracy and applications of group MEG studies using cortical source locations estimated from participants' scalp surfaces

We contend that powerful group studies can be conducted using magnetoencephalography (MEG), which can provide useful insights into the approximate distribution of the neural activity detected with MEG without requiring magnetic resonance imaging (MRI) for each participant. Instead, a participant's MRI is approximated with one chosen as a best match on the basis of the scalp surface from a database of available MRIs. Because large inter-individual variability in sulcal and gyral patterns is an inherent source of blurring in studies using grouped functional activity, the additional error introduced by this approximation procedure has little effect on the group results, and offers a sufficiently close approximation to that of the participants to yield a good indication of the true distribution of the grouped neural activity. T1-weighted MRIs of 28 adults were acquired in a variety of MR systems. An artificial functional image was prepared for each person in which eight 5 × 5 × 5 mm regions of brain activation were simulated. Spatial normalisation was applied to each image using transformations calculated using SPM99 with (1) the participant's actual MRI, and (2) the best matched MRI substituted from those of the other 27 participants. The distribution of distances between the locations of points using real and substituted MRIs had a modal value of 6 mm with 90% of cases falling below 12.5 mm. The effects of this -approach on real grouped SAM source imaging of MEG data in a verbal fluency task are also shown. The distribution of MEG activity in the estimated average response is very similar to that produced when using the real MRIs. © 2003 Wiley-Liss, Inc.

Downregulation of muscle protein degradation in sepsis by eicosapentaenoic acid (EPA)

Eicosapentaenoic acid (EPA) has been shown to attenuate muscle atrophy in cancer, starvation and hyperthermia by downregulating the increased expression of the ubiquitin-proteasome proteolytic pathway leading to a reduction in protein degradation. In the current study EPA (0.5 g/kg) administered to septic mice completely attenuated the increased protein degradation in skeletal muscle by preventing the increase in both gene expression and protein concentration of the alpha- and beta-subunits of the 20S proteasome, as well as functional activity of the proteasome, as measured by the 'chymotrypsin-like' enzyme activity. These results suggest that muscle protein catabolism in sepsis is mediated by the same intracellular signalling pathways as found in other catabolic conditions.

Mechanism of induction of muscle protein degradation by angiotensin II

Angiotensin I and II have been shown to directly induce protein degradation in skeletal muscle through an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. This investigation determines the role of the nuclear transcription factor nuclear factor-κB (NF-κB) in this process. Using murine myotubes as a surrogate model system both angiotensin I and II were found to induce activation of protein kinase C (PKC), with a parabolic dose-response curve similar to the induction of total protein degradation. Activation of PKC was required for the induction of proteasome expression, since calphostin C, a highly specific inhibitor of PKC, attenuated both the increase in total protein degradation and in proteasome expression and functional activity increased by angiotensin II. PKC is known to activate I-κB kinase (IKK), which is responsible for the phosphorylation and subsequent degradation of I-κB. Both angiotensin I and II induced an early decrease in cytoplasmic I-κB levels followed by nuclear accumulation of NF-κB. Using an NF-κB luciferase construct this was shown to increase transcriptional activation of NF-κB regulated genes. Maximal luciferase expression was seen at the same concentrations of angiotensin I/II as those inducing protein degradation. Total protein degradation induced by both angiotensin I and II was attenuated by resveratrol, which prevented nuclear accumulation of NF-κB, confirming that activation of NF-κB was responsible for the increased protein degradation. These results suggest that induction of proteasome expression by angiotensin I/II involves a signalling pathway involving PKC and NF-κB. © 2005 Elsevier Inc. All rights reserved.

Induction of protein degradation in skeletal muscle by a phorbol ester involves upregulation of the ubiquitin-proteasome proteolytic pathway

Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose-response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 μM), suggesting that it was mediated through the ubiquitin-proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin-proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome α-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E214k, also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin-proteasome pathway. TPA also induced degradation of the inhibitory protein, I-κBα, and increased nuclear accumulation of nuclear factor-κB (NF-κB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-κB activation by resveratrol (30 μM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-κB. © 2005 Elsevier Inc. All rights reserved.