Expression of the von Hippel-Lindau-binding protein-1 (Vbp1) in fetal and adult mouse tissues

BACKGROUND: Tetrahydropalmatine (THP) is a neuroactive alkaloid with analgesic and hypnotic action. Its analysis is important because cases of human poisonings have emerged as a result of unregulated use of some proprietary biopharmaceuticals containing purified THP. METHODS: We established analytical parameters for HPLC with diode-array detection (HPLC-DAD) and gas chromatography-mass spectrometry (GC-MS) for the detection of THP in serum and urine. Nine acutely THP-poisoned adults were thus screened over 16 months. RESULTS: All patients recovered quickly after mild neurological disturbance. In general, THP was metabolized rapidly and excreted as polar metabolites in urine. Serum THP was measured in five cases and found to be <0.1-1.2 mg/L (<0.3-3.4 micromol/L). Paired analyses of urine with and without glucuronidase treatment clarified the disposition of THP. Our GC-MS method with trimethylsilane derivatization identified O-desmethyl metabolites. With a uniform solid-phase extraction, the HPLC-DAD procedure detected intact glucuronide metabolites. CONCLUSION: Intact glucuronide metabolites of THP are sensitive markers for THP exposures. Our methods and findings provide practical tools and information for surveillance of intoxication caused by excessive THP intake.     

Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by competition for limiting cellular factors

Mutations in two related genes, PS1 and PS2, account for the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for "replacement" we expressed familial Alzheimer's disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s).     

Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1

Mutations in the genes encoding amyloid-beta precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2) are known to cause early-onset, autosomal dominant Alzheimer's disease. Studies of plasma and fibroblasts from subjects with these mutations have established that they all alter amyloid beta-protein (beta APP) processing, which normally leads to the secretion of amyloid-beta protein (relative molecular mass 4,000; M(r) 4K; approximately 90% A beta1-40, approximately 10% A beta1-42(43)), so that the extracellular concentration of A beta42(43) is increased. This increase in A beta42(43) is believed to be the critical change that initiates Alzheimer's disease pathogenesis because A beta42(43) is deposited early and selectively in the senile plaques that are observed in the brains of patients with all forms of the disease. To establish that the presenilin mutations increase the amount of A beta42(43) in the brain and to test whether presenilin mutations act as true (gain of function) dominants, we have now constructed mice expressing wild-type and mutant presenilin genes. Analysis of these mice showed that overexpression of mutant, but not wild-type, PS1 selectively increases brain A beta42(43). These results indicate that the presenilin mutations probably cause Alzheimer's disease through a gain of deleterious function that increases the amount of A beta42(43) in the brain.     

Biochemistry of presenilin 1

The majority of cases of early-onset familial Alzheimer disease are caused by mutations in the recently identified presenilin 1 (PS 1) gene, located on chromosome 14. PS1, a 467 amino acid protein, is predicted to be an integral membrane protein containing seven putative transmembrane domains and a large hydrophilic loop between the sixth and seventh membrane-spanning domain. We produced 7 monoclonal antibodies that react with 3 non-overlapping epitopes on the N-terminal hydrophilic tail of PS1. The monoclonal antibodies can detect the full size PS1 at M(r) 47,000 (47K) and a more abundant M(r) 28,000 (28K) product in membrane from human brain and human cell lines. We examined the sub-cellular localization by using these antibodies. Immuno-electronmicroscopic and biochemical analysis indicated that PS1 is localized on cellular membrane (plasma, endoplasmic reticulum, and perinuclear) in COS-7 cells overexpressing PS1. Interestingly, the PS1 immunoreactivity in the plasma membrane was concentrated in the regions with cell-cell contact. This observation suggests a possible role of PS1 on the cell membrane as a cell adhesion molecule. To determine the protease cleaving the full length PS1 to two fragments, we treated cells with various protease inhibitors. Only proteasome inhibitor affected the PS1 processing, indicating that proteasome is a candidate protease for PS1 proteolytic cleavage. PC12 cells transiently transfected with PS1 constructs containing different Alzheimer mutations fail to generate the 28K degradation product in contrast to PC12 cells transfected with wild type PS1. Our results indicate that missense mutations in this form of familial Alzheimer disease may act via a mechanism of impaired proteolytic processing of PS1.     

Expression of interleukin-10 in human gestational tissues

1. One way to reduce health care costs is to reduce the demand for health care services. This can be accomplished by teaching employees to make better decisions about when they should see the health care provider or go to the emergency department versus treating themselves at home using self care. 2. In an effort to reduce health care costs, a manufacturing company implemented a self care program using a publication called the HealthyLife Self Care Guide. The guide was distributed to employees during a 50 minute workshop. 3. Analysis of claims data 1 year prior to distribution of the Guide and 1 year after distribution showed a savings of $39.65 per employee (a 24.4% decrease in costs) due to reduced health care provider and emergency department visits. This amounted to a return on investment of 2.6:1. 4. It appears that implementing a self care program in a worksite setting can be an effective way to reduce employer health care costs.     

Differential expression of vasoactive intestinal peptide receptors 1 and 2 (VIP-R1 and VIP-R2) mRNA in murine lymphocytes

Vasoactive intestinal peptide (VIP), a neuropeptide present in the lymphoid microenvironment, modulates cytokine expression and affects T cell proliferation. Recent molecular studies identified two VIP receptors. VIP-R1 and VIP-R2, primarily in nonlymphoid cells. In this study, we investigate the expression of VIP-R1 and VIP-R2 mRNA in unstimulated and stimulated lymphocytes and thymocytes, and in various lymphocyte subpopulations. In contrast to VIP-R1 which is constitutively expressed, the expression of VIP-R2 is induced only following stimulation through the TCR-associated CD3 complex. Both CD4+ and CD8+ T cells express VIP-R1 and VIP-R2. Two T cell lines, EL-4.IL-2 and D10.G4.1 express exclusively VIP-R2. VIP induces the expression of the VIP-R2 gene in the absence of additional stimuli. Differential expression and regulation of the two VIP receptors in T lymphocytes suggests different physiological roles in mediating the immunomodulatory activities of VIP and related neuropeptides.     

Protein topology of presenilin 1

Mutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the "loop," N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm.     

Expression of substance P (NK1) receptor mRNA in human nose

A capillary zone electrophoretic method (CZE) was developed using an uncoated fused silica capillary for the separation and determination of the main tropane alkaloids. The applicability of the developed method for analysis of plant samples was examined by analyzing samples of transgenic Egyptian henbane Hyoscyamus muticus (L.) plants. A simple 40 mM phosphate buffer at pH 7.8 using a voltage of 20 kV was found the best for this purpose. The main tropane alkaloids, atropine and scopolamine as well as nor-(-)-scopolamine, and tropic acid, the precursor of tropane alkaloids, could be separated in less than 13 min. The linear concentration range for atropine was 5.00-140 microg ml(-1), for scopolamine 7.50-210 microg ml(-1) and for tropic acid 2.50-70.0 microg ml(-1).     

Expression of the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes

Inducible heme oxygenase (HO-1) is an antioxidant stress protein, that is mainly induced by reactive oxygen species (ROS), cytokines and hyperthermia. By using flow cytometry the present investigation demonstrated a rise in the cytoplasmic expression of HO-1 in lympho- (L), mono- (M) and granulocytes (G) of 9 endurance-trained male subjects after a half marathon run. The expression was more pronounced in M (median: 98.3% HO-1 positive cells/4.31 mfc) and G (94.8%/1.93 mfc) than in L (80.1%/1.51 mfc) when measured 3 h post-exercise. Additionally the exercise protocol caused a rise in the plasma levels of myeloperoxidase, TNF alpha and interleukin-8 (IL-8), indicating an inflammatory response. We could detect a correlation between IL-8 and HO-1, directly after exercise, that was apparent in G (r = 0.67, p < .05) and L (r = 0.80, p < .05), but did not reach significance in M (r = 0.65, p = 0.06). An additional detection of HO-1 at rest in 12 untrained subjects showed a higher baseline expression of HO-1 compared to the athletes. The regulatory pathways leading to an increased expression of HO-1 after endurance exercise are not completely clear, but a causal involvement of a cytokine-mediated generation of ROS must be discussed. We supposed that the down-regulation of the baseline expression of HO-1 in athletes reflects an adaptional mechanism to regular exercise training.     

Expression of the mnb (dyrk) protein in adult and embryonic mouse tissues

Remission of Graves' disease (GD) during pregnancy with recrudescence after delivery is commonly observed. However, as pregnancy is associated with type 2 rather than type 1 cytokine production, a decrease in thyroid-stimulating antibody (TSAb) activity alone is unlikely to account for the remission during pregnancy. We hypothesized that a change in the antibody characteristics may occur as pregnancy advances. Fifteen women were studied in the first, second, and third trimesters of pregnancy and 4 months postpartum. TSH receptor antibodies were determined using human thyroid cell cultures, and lymphocyte subsets were measured by flow cytometry. Median TSAb (determined by cAMP release) decreased from 280% (96-3200) to 130% (range, 35-350; P < 0.05) during pregnancy, but no significant change was noted with the TSH binding inhibitory antibody (TBII; determined by RRA). Thyroid stimulation-blocking antibody (TSBAb; inhibition of TSH-stimulated cAMP release) increased from 16 +/- 9% to 43 +/- 16% (mean +/- SD; P < 0.005). The increase in TSBAb was observed even among those patients who were in clinical remission before pregnancy. Overall, a negative correlation was observed between TSBAb activities and free T4 levels during pregnancy (r = -0.279; P < 0.05). Reciprocal changes in TSAb, TBII, and TSBAb levels were observed in the seven patients who relapsed during the postpartum period. In comparison, the healthy pregnant women (n = 14) were all negative for TSAb, TBII, and TSBAb throughout pregnancy. The absolute number of T lymphocytes, T helper cells, and natural killer cells, but not B cells, decreased significantly during pregnancy in both healthy women and GD patients. GD patients had significantly more CD5+ B cells at all stages of pregnancy compared to controls. In conclusion, a change in specificity from stimulatory to blocking antibodies was observed in GD patients during pregnancy and may contribute to the remission of GD during pregnancy.     

A double recombinant adenovirus expressing the costimulatory molecule B7-1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model

Tumors that express tumor-specific antigens can maintain growth in an immunocompetent organism. Current hypotheses tend toward T cell anergy as a key component for the inhibition of immunoreactivity against such tumors. Anergy is thought to occur from hyperactive stimulation of the TCR in the absence of costimulation (costimulation leads to proliferation via IL-2 production). Subcutaneous injection of transgenic polyoma middle T transformed breast adenocarcinoma tumor cells (PyMT) in the hind flank of FVB/n mice results in the formation of tumor nodules at this site. We determined the MHC class I and class II, B7-1, and B7-2 expression in the tumor cells by flow cytometry and showed positive staining for only MHC class I. We show that a single E1-deleted adenovirus constructed to express both the costimulatory molecule B7-1 (murine) and human IL-2 genes (Ad5E1 mB7-1/human IL-2) elicits a very potent antitumor response when administered intratumorally. Ad5E1 mB7-1/human IL-2 induced rapid and complete regression (100%) of all tumors compared with Ad5 E1 mB7-1 (38%), Ad CAIL-2 (42%), and Ad5E1 dl70-3 (control vector) (0%). All mice that exhibited complete tumor regression were fully protected in tumor cell challenge experiments. The systemic immunity generated by intratumoral administration of the Ad vectors was associated with a strong anti-PyMT CTL response. These observations indicate that augmenting the immunogenicity of the tumor with coincident expression of B7-1 in combination with IL-2 may prove beneficial in direct tumor immunotherapy.     

The presenilin 1 mutation (M146V) linked to familial Alzheimer's disease attenuates the neuronal differentiation of NTera 2 cells

Mutations in presenilin 1 (PS1) gene are the major cause of early-onset familial Alzheimer's disease. The biological functions of PS1 remain elusive, although accumulating evidence suggests that PS1 may play an important role in development and differentiation. To learn about the significance of PS1 in the differentiation of neuronal cells, we established NTera 2 (NT2) cell lines stably expressing wild-type (wt) or M146V mutant human PS1, and compared the differentiation of both types of cell lines into postmitotic neurons upon retinoic acid (RA) treatment. After 25 days of RA treatment, a significant proportion of cells differentiated into neurons in NT2 cells expressing wt PS1 (27.7% of total cells), which was comparable to that in untransfected cells, whereas very few cells differentiated into neurons in NT2 cells expressing M146V mutant PS1 (2.6% of total cells). These results suggest that mutant PS1 attenuates the potentials of NT2 cells to differentiate into neurons.     

Expression and localization of membrane type 1 matrix metalloproteinase in tooth tissues

Matrix metalloproteinases (MMPs) have been detected in forming dental enamel and are thought to play an important role during enamel biomineralization. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane bound member of the MMP gene family that has previously been shown to be expressed by cells associated with bone and cartilage formation (osteoclasts, osteoblasts and chondrocytes). Thus, we asked if MT1-MMP was also expressed by the cells responsible for the formation of enamel and dentin. A porcine MT1-MMP cDNA composed of 3284 bp was isolated from an enamel organ-specific cDNA library. Multiple tissue Northern blot analysis revealed that the MT1-MMP message was expressed highly in the enamel organ and pulp organ when compared to the expression levels observed in other non-mineralizing tissues. Northern blot analysis of stage-specific enamel organs (early secretory, late secretory, or maturation stage) and their corresponding pulp organs revealed that MT1-MMP expression increased as the dentin matured. In the enamel organs, however, the MT1-MMP message level became reduced only during the late secretory stage. Immunohistochemical analysis showed that MT1-MMP was present on the surface of the cells (ameloblasts and odontoblasts) responsible for dentin and enamel formation. Thus, MT1-MMP is highly expressed in developing tooth tissues and may play a role in the biomineralization of enamel and dentin.     

Interleukin-1 beta induces prostaglandin G/H synthase-2 (cyclooxygenase-2) in primary murine astrocyte cultures

Activation of glial cells and the consequent release of cytokines, proteins, and other intercellular signaling molecules is a well-recognized phenomenon in brain injury and neurodegenerative disease. We and others have previously described an inducible prostaglandin G/H synthase, known as PGHS-2 or cyclooxygenase-2, that is up-regulated in many cell systems by cytokines and growth factors and down-regulated by glucocorticoid hormones. In cultured mouse astrocytes we observed increased production of prostaglandin E2 (PGE2) after stimulation with either interleukin-1 beta (IL-1 beta) or the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). This increase in PGE2 content was blocked by pretreatment with dexamethasone and correlated with increases in cyclooxygenase activity measured at 4 h. Northern blots revealed concomitant increases in PGHS-2 mRNA levels that peaked at 2 h and were dependent on the dosage of IL-1 beta. Dexamethasone inhibited this induction of PGHS-2 mRNA by IL-1 beta. TPA, basic fibroblast growth factor, and the proinflammatory factors tumor necrosis factor alpha and lipopolysaccharide, but not interleukin-6, also stimulated PGHS-2 mRNA expression. Relative to IL-1 beta, the greater increases in PGE2 production and cyclooxygenase activity caused by TPA correlated with a greater induction of PGHS-2 mRNA. Furthermore NS-398, a specific inhibitor of cyclooxygenase-2, blocked > 80% of the cyclooxygenase activity in TPA-treated astrocytes. These findings indicate that increased expression of PGHS-2 contributes to prostaglandin production in cultured astrocytes exposed to cytokines and other factors.