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The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1 S ,3 R )-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1 H )-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [ N -(6- tert -butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a β 2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state β 2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously., (Copyright © 2018 The Author(s).)

The dopamine D(1) receptor agonist dihydrexidine (DHX) [(±)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a] phenanthridine hydrochloride] has shown efficacy in animal models of Parkinson's disease and improved cerebral blood flow and working memory of schizophrenic patients. Although the discriminative stimulus effects of DHX, an in-vivo predictor of human subjective effect profile, have only been characterized with respect to activity at D(1) receptors, DHX also has significant affinity for D(2) receptors. This study was designed to characterize the role of D(1) and D(2)/D(3) receptors in mediating the discriminative stimulus effects of DHX. Rats were trained to discriminate DHX [3 mg/kg, intraperitoneally (i.p.)] from the vehicle. The selective dopamine D(1) receptor partial agonist SKF 38393 was fully substituted for DHX. The D(1) receptor antagonist SCH 23390 (0.1 mg/kg, s.c.) and the D(3)-selective antagonist U99194 (10 mg/kg, i.p.) significantly attenuated the discriminative stimulus effects of the training dose of DHX by 80 and 60%, respectively, suggesting that both D(1) and D(3) receptors mediate the discriminative stimulus effects of DHX. In contrast, raclopride (1 mg/kg, i.p.) did not significantly alter the discriminative stimulus effects of DHX, indicating a lack of D(2)-mediated effects. The D(2)/D(3) receptor preferring agonists, quinpirole and (+)-PD 128907 were fully substituted, whereas (+)-7-OH-DPAT was partially substituted for DHX. The DHX bound to D(2) receptors with a Ki of 4.3+0.7 nmol/l was compared with 33.7+4.6 nmol/l at D(3) receptors. Determinations of activity at second messenger systems revealed that DHX functioned as a full agonist at D(3) receptors and a partial agonist at D(2) receptors in vitro. These activities at D(2)/D(3) receptors have shown effects in some preclinical models and clinical disease states. Therefore, the prominent in-vivo agonist activity of DHX at both D(1) receptors and D(2)/D(3) receptors should be considered while making predictions of effects in humans.

The hemagglutinin-tagged human trace amine-associated receptor1 (TAAR1) was stably coexpressed with rat Galpha(s) in the AV12-664 cell line, and receptor activation was measured as the stimulation of cAMP formation. After blockade of endogenously expressed alpha2- and beta-adrenoceptors with 2-[2-(2-methoxy-1,4-benzodioxanyl)]-imidazoline hydrochloride (2-methoxyidazoxan, RX821002) and alprenolol, respectively, the resulting pharmacology was consistent with that of a unique receptor subtype. beta-Phenylethylamine (beta-PEA), the putative endogenous ligand, gave an EC50 of 106 +/- 5 nM in the assay. For a series of beta-PEA analogs used to explore the pharmacophore, small substituents at ring positions 3 and/or 4 generally resulted in compounds having lower potency than beta-PEA, although several were as potent as beta-PEA. However, small substituents at ring position 2 resulted in a number of compounds having potencies as good as or better than beta-PEA. A number of nonselective antagonists known to share affinity for multiple monoaminergic receptors were evaluated for their ability to inhibit beta-PEA stimulation of the human TAAR1. None had an IC50 <10 microM. For comparison, the rat TAAR1 receptor was expressed in the AV12-664 cell line. A number of agonist compounds had significantly different relative potencies between the rat and human TAAR1, demonstrating a significant species difference between the rat and human TAAR1. The TAAR1 receptor exhibits a pharmacologic profile uniquely different from those of classic monoaminergic receptors, consistent with the structural information that places them in a distinct family of receptors. This unique pharmacologic profile suggests the potential for development of TAAR-selective agonists and antagonists to study their physiologic roles.

Beta-amyloid peptides (Abeta) are produced by a sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The lack of Abeta production in beta-APP cleaving enzyme (BACE1)(-/-) mice suggests that BACE1 is the principal beta-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1(-/-) mice supports cleavage of APP at the canonical beta-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as beta', resulting in Abeta peptides starting at Glu11. The apparent inability of human BACE1 to make this beta'-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the beta'-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the beta- and beta'-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the beta- and beta'-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated beta'-cleavage site. It does not appear to be a significant species specificity to this cleavage.

Background: Human kallikrein 7 (hK7), also known as human stratum corneum chymotryptic enzyme, is a chymotrypsin-like serine protease first identified in human skin extracts and predicted to be a secreted protease. The aim of this study was to develop a sensitive and specific immunoassay for hK7 and to examine the distribution of hK7 in tissue extracts and biological fluids., Methods: Recombinant hK7 was produced in human embryonic kidney cells (HEK293T) and purified by a three-step column chromatographic procedure. The purified hK7 was injected into mice for antibody generation. A sandwich-type immunoassay was developed with the anti-hK7 monoclonal antibodies., Results: The assay had imprecision (CV) <10% through the dynamic range of 0.2-20 microg/L and had no detectable cross-reactivity from other members in the human kallikrein gene family. Highest concentrations were found in skin, esophagus, and kidney. hK7 was also found in amniotic fluid, ascites from ovarian cancer patients, breast milk, cerebrospinal fluid, saliva, seminal plasma, serum, sweat, synovial fluid, and urine., Conclusions: This study describes the first ELISA-type immunoassay for hK7 protein quantification. hK7 is found many human tissues and in various biological fluids.

The human 5-HT(1E) receptor gene was cloned more than a decade ago. Little is known about its function, and there have been no reports of its existence in the genome of small laboratory animals. In this study, attempts to clone the 5-HT(1E) gene from the rat and mouse were unsuccessful. In fact, a search of the mouse genome database revealed that the 5-HT(1E) receptor gene is missing from the mouse genome. However, the 5-HT(1E) gene was cloned from guinea pig genomic DNA and was characterized. The guinea pig 5-HT(1E) receptor gene encodes a protein of 365 amino acids. It shares 88% (nucleic acid) and 95% (amino acid) homology with the human receptor. The guinea pig 5-HT(1E) receptor showed similar pharmacology to the human 5-HT(1E) receptor in radioligand binding assays. Serotonin (5-hydroxytryptamine, 5-HT) dose-dependently stimulated [35S]GTPgammaS binding to the guinea pig 5-HT(1E) receptor with an EC(50) of 13.6+/-1.92 nM, similar to that of the human 5-HT(1E) receptor (13.7+/-1.78 nM). Activation of the guinea pig 5-HT(1E) receptor was also achieved by ergonovine, alpha-methyl-5-HT, 1-naphthylpiperazine, methysergide, tryptamine, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Methiothepin exhibited antagonist activity. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that 5-HT(1E) mRNA was present in the guinea pig brain with the greatest abundance in the hippocampus, followed by the olfactory bulb. Lower levels were detected in the cortex, thalamus, pons, hypothalamus, midbrain, striatum, and cerebellum. Our current study marks the first identification of the 5-HT(1E) receptor gene in a commonly used laboratory animal species. This finding should allow the elucidation of the receptor's role(s) in the complex coordination of central serotonergic effects.

Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.

We have recently reported a critical role for apolipoprotein E (apoE) in the process of amyloid deposition and neuritic plaque formation in APP(V717F) transgenic (Tg) mice, an animal model of Alzheimer's disease (AD). In the present study, we have investigated whether the presence or absence of apoE alters the processing of the amyloid precursor protein (APP) to various fragments, including the beta-amyloid peptides (Abeta). Here we show that, in contrast to APP(V717F) Tg mice expressing apoE, APP(V717F) Tg mice deficient in apoE develop anti-Abeta immunoreactive multifocal aggregates, which contain the beta-cleaved C-terminal fragments (beta-CTFs) of APP. Tg mice deficient in apoE also display altered levels of mature full-length APP, increased amounts of beta-CTFs, as well as elevated levels of Abeta(1-40) and Abeta(1-42) in an age- and region-dependent manner when compared to Tg mice expressing apoE. Taken together, these data support a role for apoE in APP processing in vivo.

Background: PSA mediates growth factor responses that stimulate proliferation of prostatic and other cellular types. Androgen-sensitive TE85 human osteosarcoma cells were used to study PSA as a potential mediator of prostatic cancer growth and osseous metastasis., Materials and Methods: TE85 cells were probed for PSA mRNA and protein levels under testosterone (T)-replete and--depleted conditions. TE85 proliferative responses to PSA were evaluated in the absence and presence of LY312340, a monocyclic beta-lactam inhibitor of PSA enzymatic activity., Results: A 3.1-fold increase in PSA mRNA was observed following T stimulation. Low levels of immunoreactive PSA (iPSA) were detected in media of androgen-stimulated TE85 cells while iPSA was not found in control media. Conversely, iPSA was detected in TE85 cell pellets from control but not in androgen-stimulated cell cultures. Exogenously added enzymatically active PSA stimulated TE85 proliferation in a bi-phasic manner. LY312340 inhibited PSA-induced increases in TE85 cell numbers but had no effect on basal or T- stimulated cellular proliferation., Conclusion: While the PSA levels produced by TE-85 cells in response to androgen stimulation are too low to be biologically active, PSA produced by metastatic PCa cells may mediate paracrine stimulation of osteogenic PCa metastasis. Inhibitors of PSA enzymatic activity could be useful therapeutic agents.

The KLK6 gene is a new member of the human kallikrein gene family and encodes for a secreted protease, human kallikrein 6 (hK6; also known as zyme/protease M/neurosin). No study has as yet reported detailed immunohistochemical localization of hK6 in human tissues. Our purpose was to examine the expression of hK6 in human tissues by immunohistochemistry. We have analyzed 199 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue. We employed an hK6-specific polyclonal rabbit antibody and avidin-biotin to localize hK6 by IHC. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. The IHC expression of zyme was generally cytoplasmic. Various normal human tissues expressed the protein abundantly. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, endometrium, colon, appendix, salivary glands, bile ducts, and gallbladder. The small intestine, stomach, endocervix, Fallopian tube, epididymis, bronchus, and upper respiratory tract showed a focal expression as well. Choroid plexus epithelium, peripheral nerves, and some neuroendocrine cells (including the islets of Langerhans, cells in the anterior pituitary gland, and adrenal medulla) expressed the protein strongly and diffusely. A characteristic immunostaining was observed in the Hassall's corpuscles of the thymus, the oxyphilic cells of the thyroid and parathyroid glands, the primordial follicles of the ovary, dendritic cells mainly in the spleen, and in various cells of the placenta.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

Secreted isoforms of the beta-amyloid precursor protein potently enhance neuronal survival in cell cultures exposed to toxic amyloid beta peptide. Lowering of intracellular calcium levels to offset the increases in intraneuronal calcium caused by amyloid beta peptide is thought to underly this neuroprotection. Because we have shown previously that an amyloid beta peptide-mediated potentiation of calcium channel currents may contribute to this cytosolic calcium overload, the present study examined the effects of a secreted beta-amyloid precursor protein on the calcium channel response to amyloid beta peptide. When compared with untreated cultured rat hippocampal neurons, cells that underwent a 24 h preincubation with beta-amyloid precursor protein 751 displayed decreases in the relative size of the calcium channel response to amyloid beta peptide. A membrane-permeable analog of cyclic GMP, a second messenger believed to be involved in the calcium regulation process mediated by beta-amyloid precursor proteins, also attenuated the modulatory calcium channel response. Co-application of beta-amyloid precursor protein 751 with amyloid beta peptide did not alter calcium channel response to amyloid beta peptide. Taken together, these findings suggest that secreted beta-amyloid precursor proteins can suppress a calcium channel response to amyloid beta peptide that is potentially injurious to the cell, and as such, may define a neuroprotective mechanism that is specific for amyloid beta toxicity.

Global brain ischemia causes cell death in the CA1 region of the hippocampus 3-5 days after reperfusion. The biological pathway leading to such delayed neuronal damage has not been established. By using differential display analysis, we examined expression levels of poly(A) RNAs isolated from hippocampal extracts prepared from rats exposed to global ischemia and found an up-regulated transcript, clone 17a. Northern blot analysis of clone 17a showed an approximately 35-fold increase in the ischemic brain at 24 h after four-vessel occlusion. Rapid amplification of cDNA ends of clone 17a revealed a family of genes (160-540 base pairs) that had the characteristics of rodent B(2) sequences. In situ hybridization demonstrated that the elevated expression of this gene was localized predominantly in the CA1 pyramidal neurons. The level of expression in the CA1 region decreased dramatically between 24 and 72 h after ischemia. The elevated expression of clone 17a was not observed in four-vessel occlusion rats treated with the compound LY231617, an antioxidant known to exert neuroprotection in rats subjected to global ischemia. Since delayed neuronal death has the characteristics of apoptosis, we speculate that clone 17a may be involved in apoptosis. We examined the expression level of clone 17a in in vitro models of apoptosis using cerebellar granule neurons that were subjected to potassium removal, glutamate toxicity, or 6-hydroxydopamine treatment and found that clone 17a transcripts were induced in cerebellar granule neurons by glutamate or 6-hydroxydopamine stimulation but not potassium withdrawal.

Background and Purpose: Nuclear factor-kappaB (NF-kappaB) is an oxidative stress responsive transcription factor that is transiently activated in most forebrain neurons in response to transient global ischemia. However, in hippocampal CA1 neurons destined to die, NF-kappaB remains persistently activated. The present study was performed to determine whether an antioxidant (LY231617) that afforded neuroprotection in previous studies had any effect on NF-kappaB activation in hippocampal CA1 neurons after global ischemia., Methods: Rats were subjected to 30 minutes of forebrain ischemia by 4-vessel occlusion (4-VO) and killed at 24 and 72 hours after ischemia. LY231617 was administered orally at a dose of 50 mg/kg 30 minutes before 4-VO and again 4 hours after 4-VO. Neuronal damage was evaluated in sections stained with cresyl violet. Other sections were immunostained with antibodies to NF-kappaB p50 to assess nuclear localization. An electrophoretic mobility shift assay was performed on nuclear extracts from sham- and LY231617-treated rats at 24 and 72 hours after ischemia., Results: The administration of LY231617 had a significant protective effect on hippocampal CA1 neurons at 72 hours after ischemia (control group, 16 +/- 7 neurons/mm; treated group, 294 +/- 35 neurons/mm, P<.02) and prevented nuclear translocation of activated NF-kappaB as normally seen at 72 hours after ischemia in untreated controls. In contrast, the untreated controls showed activated NF-kappaB at 72 hours after ischemia. At 24 hours after ischemia, both the control group and the LY231617 group showed intense nuclear localization of NF-kappaB., Conclusions: Activation of NF-kappaB in vitro has been reported to promote proapoptotic as well as antiapoptotic mechanisms, depending on the cell type being investigated. In the present in vivo study, the role of the transient activation of NF-kappaB observed at 24 hours may be responsible for the induction of protective factors in neurons that survive the ischemic insult, whereas the persistent activation of NF-kappaB in hippocampal neurons could be responsible for the induction of proteins that result in CA1 neuronal death.

Beta-amyloid peptides (A beta) are deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of A beta in cultured neurons is dependent on its aggregation state, but the factors contributing to aggregation and fibril formation are poorly understood. In the present study, we investigated whether alpha2-macroglobulin (alpha2M), a protein present in neuritic plaques and elevated in Alzheimer's disease brain, is a potential regulatory factor for A beta fibril formation. Previous studies in our laboratory have shown that alpha2M is an A beta binding protein. We now report that, in contrast to another plaque-associated protein, alpha1-antichymotrypsin, alpha2M coincubated with A beta significantly reduces aggregation and fibril formation in vitro. Additionally, cultured fetal rat cortical neurons are less vulnerable to the toxic actions of aged A beta following pretreatment with alpha2M. We postulate that alpha2M is able to maintain A beta in a soluble state, preventing fibril formation and associated neurotoxicity.

Transgenic animals were used to examine the spatial and temporal regulation of the human beta amyloid precursor protein (APP) gene promoter region in vivo. A 2.9 kb DNA fragment encompassing the APP gene promoter was fused to the chloramphenical acetyltransferase (CAT) reporter gene (pAMY-CAT) or a partial cDNA encoding the potentially amyloidogenic C-terminal 100 amino acid region of APP (pAMY-C100). Expression of these transgenes occurred primarily, but not exclusively, in the central nervous system (CNS) and testis in multiple independent lineages of transgenic mice. Temporal expression of the CAT reporter gene during development paralleled that reported for the endogenous APP gene. These studies suggest that a CNS-responsive cis-acting element(s) may exist in the promoter/5'-flanking region of the APP gene.

Hippocampal CA1 neurons are highly susceptible to short periods of transient global ischemia. We have previously reported in a rat model of transient forebrain global ischemia that activation and nuclear localization of NF-kB occurs in the CA1 neurons at 24 and 72 h post reperfusion. Events following NF-kB activation would ultimately determine whether damaged cells will undergo programmed cell death. We have selected bcl-x gene expression for study because there is increasing evidence that proteins encoded by the bcl-2 gene family (bcl-2, bcl-x, bax etc) play a role in the regulation of programmed cell death. We have observed that the bcl-x gene promoter contains a putative consensus sequence for NF-kB/CS4 responsive activation. We also can show that other members of the bcl-2 multigene family contain the NF-kB/CS4 sequence in their five prime regulatory regions. In this study, we show that NF-kB p50 and NF-kB p65 act in synergy to transactivate the bcl-x promoter in co-transfected 293 cells. We also report that following ischemia and NF-kB activation, bcl-x messenger RNA levels increase in the CA1 hippocampal region. As a result of this transcriptional increase, surprisingly, it is bcl-xs, the apoptotic form of bcl-x, that is elevated. These results suggest that activation of NF-kB can lead to increased expression of bcl-x as manifested by the increase in the short form of bcl-x.

The deposition of the beta amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid beta-protein of 39-43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP. We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease. When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid beta-protein peptide and shows a reduction of residues 17-42 of Abeta (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.

The oxidative stress responsive transcription factor nuclear factor-kappa B (NF-kappa B) consists of a p50 (50 kDa) and p65/RelA (65 kDa) component and can be activated in vitro by TNF alpha, IL1 beta, hydrogen peroxide and oxygen radicals. All of the above factors are also known to be elevated at certain times after transient global ischemia. The present study was performed to determine if NF-kappa B was activated in vivo by transient global forebrain ischemia. Adult male rats were subjected to 30 min of 4-vessel occlusion (4-VO) and sacrificed at selected post-ischemic time points. Levels of NF-kappa B p50 and p65 subunits were determined by immunocytochemistry, Western blot and electrophoretic mobility-shift analysis. The enhancer complex was also confirmed by immuno-gel-shift analysis. Specific labeling of DNA strand breaks and DNA fragmentation was examined in situ by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Western blot analysis of hippocampus showed induction of p50 and p65. A time course of NF-kappa B induction in hippocampus showed a p50-specific band at 6 h that increased in intensity over 12, 48 h and then decreased by 96 h post-ischemia. Immunocytochemistry revealed at 24 h post-ischemia that p65 and p50 immunoreactivity was present in neuronal nuclei of hippocampal CA1 neurons as well as all other hippocampal regions and several other forebrain regions which were not vulnerable to transient forebrain ischemia. At 72 h post-ischemia, nuclear NF-kappa B immunoreactivity had disappeared in all brain areas except in hippocampal CA1 neurons which were degenerating. No evidence for DNA fragmentation as revealed by TUNEL staining could be observed at 24 h. However, at 72 h, hippocampal CA1 neurons were heavily labeled. The results of this study demonstrate that global forebrain ischemia causes a transient activation of NF-kappa B in many forebrain regions. NF-kappa B remains persistently activated in the vulnerable hippocampal CA1 sector. Because of the persistent activation of NF-kappa B in these neurons, the possibility exists that NF-kappa B has a role in programmed cell death in hippocampal CA1 neurons.

Thrombin, an important mitogen governing smooth muscle cell proliferation, binds to cultured bovine aortic smooth muscle cells (BASMCs) via both the proteolytically activated thrombin receptor (PATR) and thrombomodulin (TM). Although TM mRNA expression and functional activity is regulated by thrombin in human endothelial cells and mouse hemangioma cells, it remains unclear in those models whether the increased TM mRNA expression observed upon thrombin stimulation is mediated through the activation of PATR or via TM occupancy. We observed in cultured BASMCs that TM mRNA is increased threefold to sixfold by either thrombin, basic fibroblast growth factor (bFGF), or platelet-derived growth factor (PDGF). The increase in TM mRNA with thrombin is time dependent (maximal at 3 hours), a consequence of increased mRNA stability, and accompanied by increases in cell surface TM functional activity. Thrombin-induced TM mRNA was reproduced by the hexameric thrombin receptor-activating peptide (TRAP6) and augmented by a TM-specific antibody. Together, these data suggest that up-regulation of TM mRNA by thrombin is mediated via the PATR. We speculate that increases in BASMC TM mRNA and activity after thrombin may contribute to the impaired thrombus formation observed after atherosclerotic vascular injury.

Background and Purpose: After global ischemia, brain levels of hydrogen peroxide, oxygen radicals, and the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are increased. Oxygen radicals, TNF-alpha, and IL-1 beta are known to activate nuclear factor-kappa B (NF-kappa B) in vitro. The present study was performed to determine whether NF-kappa B was activated in vivo by global ischemia in hippocampal CA1 neurons., Methods: Adult male rats were subjected to 30 minutes of four-vessel occlusion and killed 72 hours later. Levels of NF-kappa B p50 and p65 subunits in hippocampus were determined by immunocytochemistry, Western blot, and gel-shift analysis. Specific labeling of DNA strand breaks was demonstrated by means of an Apoptag apoptosis detection kit., Results: Labeling of DNA strand breaks was present at 72 hours. Chromatin compaction and segregation, a characteristic of apoptosis, was observed in sections stained with hematoxylin and eosin. NF-kappa B p50 and p65 immunoreactivity localized only to nuclei of CA1 neurons at 72 hours after reperfusion. Induction of the activated p50 and p65 subunits was confirmed by Western blot and electromobility shift analysis. The results demonstrate that NF-kappa B is activated selectively in hippocampal CA1 neurons at 72 hours after four-vessel occlusion, which is at the approximate time of CA1 neuronal cell death., Conclusions: Transient forebrain ischemia resulted in a marked activation of nuclear NF-kappa B in the highly vulnerable CA1 sector. Intense nuclear localization of NF-kappa B was associated only with dying neurons; regions of the hippocampus that were not vulnerable to four-vessel occlusion did not exhibit nuclear NF-kappa B localization. The elevation of NF-kappa B in degenerating CA1 neurons may be associated mechanistically with apoptotic or necrotic cell death.

Thrombin and other mitogens regulate the expression of the urokinase-type plasminogen activator receptor (uPAR) protein and mRNA levels in bovine vascular smooth muscle cells (SMC). We investigated interactions between mitogens capable of increasing uPAR mRNA levels in SMC. Up-regulation of uPAR mRNA upon thrombin and basic fibroblast growth factor (bFGF) stimulation was preceded by a 2-3-fold transient increase in bFGF mRNA within 1 h. TGF-beta1 did not result in a significant change in bFGF mRNA levels. Platelet-derived growth factor (PDGF) while substantially enhancing uPAR mRNA levels, diminished bFGF mRNA levels by 3-4-fold. Both thrombin and bFGF induced the message for bFGF-R 2-3-fold. Thrombin also provoked a 3-4-fold rise in TGF-beta1 mRNA levels within 30 min. In summary, on the mRNA level, we demonstrated both positive as well as negative feed-back mechanisms between different mitogens, among them bFGF revealing in addition to autoinduction also up-regulation of the transcript concentration of its own receptor. Thus, cooperation and possible amplification of mitogenic effects might be implicated in the fine-tuned regulation of uPAR mRNA in stimulated bovine aorta SMC.

Cultures of transformed human embryonic kidney 293 cells were transiently transfected with minigene constructs coding for the Abeta peptide (1-43). The Abeta minigene used in this study consisted of exons 16 and 17 of the amyloid precursor protein gene, including the 6000+ bp intronic region. Two of the constructs used in this study, human amyloid precursor protein (APP) promoter-driven Abeta minigene and BK virus enhancer/adenovirus major late promoter-driven Abeta minigene, did not contain a signal peptide sequence, whereas the third, human APP promoter-signal peptide Abeta minigene did not contain the human APP signal sequence. The resulting Abeta products were detected by immune precipitation, using 10D5 antibody and Western blot analysis, using R1280 antisera, as SDS stable oligomers in cell lysates of cells containing all three constructs or in culture media when produced by the signal peptide construct. Evaluation of the cells by immunocytochemistry using conventional and transmission electron microscopy indicated that the cells transfected with constructs without the signal peptide accumulated immunoreactive Abeta primarily in the nucleus.

Background and Purpose: Phospholipid breakdown has been reported to be an early event in the brain after global cerebral ischemia. Our earlier observations showing the localization of cytosolic phospholipase A2 (cPLA2) to astrocytes in aged human brains and the intense glial activation observed after global forebrain ischemia prompted us to investigate the cellular localization of cPLA2 in the rat brain subjected to global ischemia., Methods: Immunohistochemistry was performed in sections through the dorsal hippocampus in rats subjected to 30 minutes of four- vessel occlusion. PLA2 was localized with the use of a highly selective antiserum. Double immunofluorescent localization was performed to colocalize cPLA2 with various glial cell types. cPLA2 levels were also measured by enzymatic assay and Western blot analysis., Results: A marked induction of cPLA2 was observed in activated microglia and astrocytes in the CA1 hippocampal region at 72 hours after ischemia. Only a subset of astrocytes and microglia were immunoreactive for cPLA2. Twenty-four hours after ischemia, numerous cPLA2 immunoreactive astrocytes were observed. Western blot analysis of hippocampal homogenates at 72 hours after ischemia showed induction of a 100-kD band that comigrated with purified human cPLA2, and a threefold induction in cPLA2 activity was demonstrated by enzymatic assay., Conclusions: These results indicate that both reactive astrocytes and microglia contain elevated levels of cPLA2. Induction of cPLA2 was confined to areas of neurodegeneration and likely precedes its onset. The results suggest that reactive glia may play a role in the pathophysiology of delayed neuronal death after transient global forebrain ischemia.

The expression and localization of tensin and cortactin were examined in osteoclast precursors in comparison with isolated osteoclasts on various substrates. Initially, the ability of hen monocytes to differentiate into osteoclasts was evaluated on plastic or glass, and compared to differentiation on bone. Specifically, monocytes were isolated from the medullary bones of egg-laying hens maintained on a Ca-deficient diet. Differentiation was monitored morphologically and by quantitation of the ability to form Howship's lacunae in bone slices or resorb radiolabeled bone particles of 20-53 microns diameter. These cells differentiated into tartrate resistant acid phosphatase (TRAP)-positive, bone resorbing, multinucleated syncytia in the presence of cytosine-1-beta-D-arabinofuranoside in a time dependent manner (day 1-6). Differentiation into osteoclast-like cells was similar whether cultured on plastic, on glass, or on bone. When compared to GAP-DH control levels, tensin and cortactin mRNA levels increased by 7- and 10-fold, respectively, by day 6. Tensin and cortactin protein levels also increased by 6- and 15-fold, respectively, by day 6. Immunofluorescence of differentiating precursors showed that tensin localized between regions of cell to cell contact and colocalized with vinculin in podosomes of osteoclast-like cells and of real osteoclasts. Cortactin immunofluorescence was not detectable in monocytes but localized inside tensin/vinculin podosome structures after fusion into osteoclast-like cells and in freshly isolated osteoclasts. Both tensin and cortactin were associated with attachment complexes used by osteoclast-like cells and osteoclasts to resorb bone. Specifically, punctate cortactin staining was observed inside tensin staining which formed a double ring structure at the membrane/bone interface of resorbing osteoclasts. These data showed that tensin and cortactin can be used as osteoclast differentiation markers, that participate in attachment complexes used to resorb bone, and that tensin may participate in the fusion process of osteoclast precursors.

The expression of the carboxyl-terminal 100 (C-100) residues of the amyloid precursor protein (APP) may provide a model for studying the processing of APP to the 42-43 residue beta-amyloid peptide (beta A4) implicated in Alzheimer's disease. Expression of human C-100 in mammalian cells reportedly causes 'toxicity' and amyloid-like fibrils. We have expressed the C-100 fragment in human embryonic kidney cells (293 cells) in a transient assay and compared it to the expression of transfected wild type and mutant (Swedish familial Alzheimer's disease) full length APP. Products were characterized by Western blot analysis using antibodies to the carboxyl-terminal region of APP.

A peptide corresponding to the amino acid sequence of A beta 17-42 (LVFFAEDVGSNKGAIIGLMVGGVVIA) was isolated from Alzheimer Disease patient brains containing large deposits of diffuse-type amyloid. Brain homogenates were lysed in SDS and submitted to high speed centrifugations. A beta peptides were purified by size exclusion chromatography on Superose 12 and TSK 3000 SW columns. An A beta peptide with M(r) of 3,000 was recovered that on automatic gas-phase Edman degradation yielded the amino acid sequence of A beta starting at residue 17 (Leu). The 3-kDa peptide was subsequently hydrolyzed with trypsin and reacted with CNBr, and the resulting peptides were separated by reverse phase high pressure liquid chromatography and characterized by amino acid analyses, peptide microsequencing, and mass spectrometry. Hydrolysis of beta-amyloid precursor protein 695 at Lys612-Leu613 or at Lys16-Leu17 of its A beta 1-42 derivative prevents the generation of neurotoxic A beta filaments, thus leading to the formation of A beta 17-42 localized in the diffuse amyloid deposits. An outstanding feature in the pathology of Alzheimer Disease is that the predominant A beta peptides have their C termini at position 42, whether in the cores of the neuritic plaques, in the vascular walls, or in the diffuse deposits. Based on these observations, we postulate that the accumulation of insoluble A beta N-42 in Alzheimer Disease is due to the anomalous processing of the C-terminal region.

We isolated a full length cDNA clone for bovine u-PAR from a bovine aorta endothelial cell cDNA library and compared the structural properties of this receptor protein to the published human and murine sequences. The bovine u-PAR cDNA clone spans a nucleotide sequence stretch of 1335 bp. The open reading frame contains 330 amino acids with a 20 amino acid long putative signal peptide. The mature protein contains 6 potential N-linked glycosylation sites and a high cysteine content (9%). Bovine u-PAR revealed three homologous internal structural repeats. The NH2-terminal repeat containing the u-PA binding site showed 54% identity to the human and murine NH2-terminal domain, compared to 64% identity between human and mouse u-PAR. Southern blot analysis of genomic DNAs from 9 eukaryotic species suggests that the u-PAR gene is conserved in man, monkey, and cow.

We report the novel synthesis of polymerase chain reaction (PCR)-derived single-stranded DNA (ssDNA) probes and their subsequent application in in situ hybridizations. Serial transverse sections of an 11.5-day postcoitum mouse embryo were hybridized to a 33P-ssDNA, 33P-RNA, or 35S-RNA probe corresponding to the same 181-bp sequence in the myogenin cDNA. Signal obtained using 33P-ssDNA was more intense than that using 33P-RNA probe, while signal/noise ratios obtained with both 33P-probes were far superior to those obtained with 35S-probe. Digoxigenin-labeled chicken growth hormone (GH) ssDNA gave slightly more intense signal than did digoxigenin-labeled chicken GH RNA when hybridized to chicken pituitary sections. 32P-ssDNA probes were found to be suitable for Northern blot hybridization. Advantages of using ssDNA probes for in situ hybridization include: (1) The ssDNA technique is rapid and simple. There was no need to clone a DNA template into a special RNA vector or order special T7-containing PCR primers. ssDNA probes can be synthesized in less than 1 day using any primers which currently exist in a laboratory (optimal probe length for in situ hybridization is between 50 and 200 bp). (2) In three separate in situ experiments, ssDNA probes yielded more intense signal than RNA probes. (3) ssDNA probes are potentially more stable than RNA probes. (4) Since the RNAse rinse is eliminated, posthybridization rinses are shortened when hybridizing with ssDNA probes. The ssDNA probes produced by this protocol can be labeled with a variety of different isotopes (both radioactive and nonradioactive), and are excellent probes for use in in situ hybridizations.

A recombinant infectious bovine rhinotracheitis virus (IBRV) vector has been constructed to express bovine growth hormone signal sequence plus a foot-and-mouth disease virus [FMDV (O1K)] capsid protein (VP1) epitope as the N-terminal sequence of an IBRV glycoprotein gIII fusion protein on the surface of virus infected cells and on the surface of virus particles. Sequences encoding the first 38 amino acids of IBRV gIII were deleted from the recombinant to avoid redundant glycoprotein signal sequences, but IBRV gIII epitopes detected by anti-gIII monoclonal antibodies were retained. Phenotypes were confirmed by in situ immunostaining of virus plaques with anti-FMDV peptide sera, by immunogold staining of permeabilized- and non-permeabilized infected cells, and by virus neutralization experiments with anti-FMDV peptide sera. Vaccination with the IBRV-FMDV recombinant induced protective levels of anti-FMDV antibodies in calves and protected them from challenge with virulent IBRV.

Neuritic plaque and cerebrovascular amyloid deposits have been detected in the aged monkey, dog, and polar bear and have rarely been found in aged rodents (Biochem. Biophy. Res. Commun., 12 (1984) 885-890; Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 4245-4249). To determine if the primary structure of the 42-43 residue amyloid peptide is conserved in species that accumulate plaques, the region of the amyloid precursor protein (APP) cDNA that encodes the peptide region was amplified by the polymerase chain reaction and sequenced. The deduced amino acid sequence was compared to those species where amyloid accumulation has not been detected. The DNA sequences of dog, polar bear, rabbit, cow, sheep, pig and guinea pig were compared and a phylogenetic tree was generated. We conclude that the amino acid sequence of dog and polar bear and other mammals which may form amyloid plaques is conserved and the species where amyloid has not been detected (mouse, rat) may be evolutionarily a distinct group. In addition, the predicted secondary structure of mouse and rat amyloid that differs from that of amyloid bearing species is its lack of propensity to form a beta sheeted structure. Thus, a cross-species examination of the amyloid peptide may suggest what is essential for amyloid deposition.

Modified-live, attenuated infectious bovine rhinotracheitis (IBR) hybrid virus vaccines have been constructed by inserting in the major IBRV glycoprotein gIII gene chemically synthesized deoxyribonucleotide sequences encoding the bovine growth hormone signal sequence and monomeric or dimeric forms of the foot and mouth disease virus (FMDV) VP 1 epitope sequences. The foreign DNA sequences were inserted at the N-terminal end of the IBRV gIII coding sequence and were driven by the IBRV gIII promoter. The sequences encoding the first 38 and the first 21 amino acids of the IBRV gIII were deleted from the hybrid viruses containing inserts of the monomeric and dimeric FMDV epitope sequences, respectively, to avoid redundant signal sequences. Plaque immunoassay experiments with guinea pig and bovine anti-FMDV peptide antisera, and with anti-IBRV gIII monoclonal antibodies demonstrated that IBRV-FMDV fusion proteins were expressed in virus-infected MDBK cells. Immunoelectron microscopy analyses demonstrated that the IBRV-FMDV fusion proteins were expressed as repeated structures on the surface of virus particles. Experiments showed that the recombinant IBRV-FMDV viruses protected cattle from IBRV (Cooper) challenge and induced anti-FMDV peptide antibodies, thereby demonstrating that the FMDV epitopes were expressed in vivo.

To better understand the processing of the Alzheimer disease amyloid precursor protein, we have cloned and sequenced that region of the human genome coding for the amyloid peptide. Two exons separated by a 6.2kb intron define this region. Characterization of the A4 peptide amino acid sequence shows similarity to the structure of soybean trypsin inhibitor (Kunitz). Our observation describes a different region of PreA4 than the previously characterized domain of larger amyloid precursor molecules PreA4 751 and 770(2). Moreover, the exon organization, Kunitz domain duplication and transmembrane location of A4 suggest that PreA4 is similar to growth factor precursors and thus may be processed similarly.

In a culture of Chinese hamster ovary cells infected with vesicular stomatitis virus, there is specific shedding of viral antigens into the medium. This shedding appears to be unrelated to progeny formation or to cell lysis. Although all five of the virus-specific proteins are detected in the extracellular soluble fraction, the major antigen is the Gs protein. This protein has a molecular weight of 54,000. Indirect analysis of the content of sialic acid as well as peptide analysis of the Gs and G proteins of vesicular stomatitis virus suggest that the Gs protein is derived from the G protein by proteolysis. Both proteins are hydrophobic when analyzed by charge-shift electrophoresis. The presence of phenylmethylsulfonyl fluoride in the culture medium or the removal of serum from the culture medium partially reduces the shedding of Gs protein. Increased shedding of the Gs protein is seen when there is an unstable M or matrix protein synthesized by a temperature-sensitive mutant, tsG31. These results indicate that the G protein is cleaved at the cell surface, thus releasing Gs protein into the medium. Furthermore, the stability of G protein at the cell surface appears to be dependent on its association with the M protein.

In order to evaluate the importance of the carbohydrate moiety of human tissue plasminogen activator (TPA), human melanoma (Bowes) cells were treated with a glycosylation inhibitor, tunicamycin (TM), and cellular fractions were assayed for fibrinolytic activity. Where glycosylation was inhibited by 90% and protein synthesis by 30%, TPA specific activity measured by fibrinolytic assays decreased 6-10-fold in the tissue culture medium and cell cytosol with a concomitant 2-fold increase in the 100000g microsomal pellet. In addition, TPA purified to apparent homogeneity was treated with endo-beta-N-acetylglucosaminidase H (Endo-H), producing a fraction that in contrast to native TPA did not adsorb to concanavalin A-Sepharose (Con A-Sepharose). This fraction represented TPA from which 85-90% of N-linked carbohydrate residues had been removed. Native TPA effectively activated plasminogen in the presence of fibrin (Km = 1 microM, kcat = 0.09 s-1) whereas saturation of the enzyme was not achieved at 100 microM plasminogen in the absence of fibrin. Glycosidase-treated and native TPA activated plasminogen at identical high rates in the presence and at identical negligible rates in the absence of fibrin. These studies indicate that the inhibition of glycosylation of TPA results in the inhibition of secretion of the molecule as has been observed for some other glycoproteins. The enzymatic removal of N-linked carbohydrate from purified TPA does not change its unique fibrin-directed properties.

Temperature-sensitive mutants of vesicular stomatitis virus (VSV) belonging to complementation group III contain a lesion in the matrix (M) protein. This results in a 2--5 fold increase in transcription at the nonpermissive temperature. Co-infection of cells with one of these mutants and wild-type virus reverses this mutant phenotype. Separation of the transcriptional and translational products from mutant-infected cells reveals an overall increase in each of the viral mRNA species concomitant with degradation of the M protein at the nonpermissive temperature. The increase in mRNA, however, does not lead to increased synthesis of viral proteins. Quantitation of individual mRNA species indicates that M protein acts as a direct inhibitor of transcription as well as an attenuator of sequential transcription.