Toward early estimation and treatment of addiction vulnerability: radial arm maze and N-acetyl cysteine before cocaine sensitization or nicotine self-administration in neonatal ventral hippocampal lesion rats.

RATIONAL: Prefrontal cortical (PFC)-hippocampal-striatal circuits, interconnected via glutamatergic signaling, are dysfunctional in mental illnesses that involve addiction vulnerability. OBJECTIVES: In healthy and neurodevelopmentally altered rats, we examined how Radial Arm Maze (RAM) performance estimates addiction vulnerability, and how starting a glutamatergic modulating agent, N-acetyl cysteine (NAC) in adolescence alters adult mental illness and/or addiction phenotypes. METHODS: Rats with neonatal ventral hippocampal lesions (NVHL) vs. SHAM-operated controls were randomized to NAC vs. saline in adolescence followed by cognitive testing (RAM) in early adulthood and then cocaine behavioral sensitization (experiment 1; n = 80) or nicotine self-administration (experiment 2; n = 12). RESULTS: In experiment 1, NVHL rats showed over-consumption of food (Froot-Loops (FL)) baiting the RAM with poor working memory (low-arm entries to repeat (ETR)), producing an elevated FL to ETR ratio ("FLETR"; p < 0.001). FLETR was the best linear estimator (compared to FL or ETR) of magnitude of long-term cocaine sensitization (R 2 = 0.14, p < 0.001). NAC treatment did not alter FL, ETR, FLETR, or cocaine sensitization. In experiment 2, FLETR also significantly and uniquely correlated with subsequent drug seeking during nicotine-induced reinstatement after extinction of nicotine self-administration (R 2 = 0.47, p < 0.01). NAC did not alter RAM performance, but significantly reversed NVHL-induced increases in nicotine seeking during extinction and reinstatement. CONCLUSIONS: These findings demonstrate the utility of animal models of mental illness with addiction vulnerability for developing novel diagnostic measures of PFC-hippocampal-striatal circuit dysfunction that may reflect addiction risk. Such tests may direct pharmacological treatments prior to adulthood and addictive drug exposure, to prevent or treat adult addictions.

An adenovirus-simian immunodeficiency virus env vaccine elicits humoral, cellular, and mucosal immune responses in rhesus macaques and decreases viral burden following vaginal challenge.

Six female rhesus macaques were immunized orally and intranasally at 0 weeks and intratracheally at 12 weeks with an adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus SIVsm env recombinant and at 24 and 36 weeks with native SIVmac251 gp120 in Syntex adjuvant. Four macaques received the Ad5hr vector and adjuvant alone; two additional controls were naive. In vivo replication of the Ad5hr wild-type and recombinant vectors occurred with detection of Ad5 DNA in stool samples and/or nasal secretions in all macaques and increases in Ad5 neutralizing antibody in 9 of 10 macaques following Ad administrations. SIV-specific neutralizing antibodies appeared after the second recombinant immunization and rose to titers > 10,000 following the second subunit boost. Immunoglobulin G (IgG) and IgA antibodies able to bind gp120 developed in nasal and rectal secretions, and SIV-specific IgGs were also observed in vaginal secretions and saliva. T-cell proliferative responses to SIV gp140 and T-helper epitopes were sporadically detected in all immunized macaques. Following vaginal challenge with SIVmac251, transient or persistent infection resulted in both immunized and control monkeys. The mean viral burden in persistently infected immunized macaques was significantly decreased in the primary infection period compared to that of control macaques. These results establish in vivo use of the Ad5hr vector, which overcomes the host range restriction of human Ads for rhesus macaques, thereby providing a new model for evaluation of Ad-based vaccines. In addition, they show that a vaccine regimen using the Ad5hr-SIV env recombinant and gp120 subunit induces strong humoral, cellular, and mucosal immunity in rhesus macaques. The reduced viral burden achieved solely with an env-based vaccine supports further development of Ad-based vaccines comprising additional viral components for immune therapy and AIDS vaccine development.

Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Immunogenicity of recombinant influenza virus haemagglutinin carrying peptides from the envelope protein of human immunodeficiency virus type 1.

Haemagglutinin (HA), the major surface glycoprotein of influenza virus, is a potent immunogen against which viral neutralizing antibodies are directed. Studies of the three-dimensional structure of HA have identified major antigenic sites on the molecule. We have exploited HA as a carrier for small antigenic regions (epitopes) of the HIV-1 envelope (env) glycoprotein. Using recombinant DNA techniques, the epitopes were inserted in-frame into a known antigenic site of HA to produce HA-epitope chimeras. Guinea-pigs and mice immunized with these chimeras in combination with adjuvant generated significant immune responses against the carrier HA and also produced epitope-specific antibodies that recognized the native whole HIV-1 env. One of the chimeras which contained a V3-loop sequence of HIV-1 env elicited neutralizing antibodies against the homologous strain of HIV-1. The antibodies against HA and the inserted epitopes remained at high levels for up to 72 weeks. Remarkably, these responses were generated with low doses of immunogens containing only nanogram quantities of the inserted epitopes. These results suggest the utility of HA as a carrier to allow selective antibody induction against foreign epitopes, and offer a new approach for vaccine development as well as for the production of monospecific antibodies useful in diagnostics and research.

Ultrastructural characterization of human immunodeficiency virus type 1 Gag-containing particles assembled in a recombinant adenovirus vector system.

The human immunodeficiency virus type 1 (HIV-1) Gag protein was expressed in A549 cells infected with recombinant adenovirus types 4 and 7, each carrying the HIV-1 gag and pro genes. The Gag protein was assembled into enveloped virus-like particles that budded from plasma and vacuolar membranes. The particles, isolated by precipitation and isopycnic density centrifugation, contained both processed and unprocessed Gag-associated proteins.

Induction of sustained patency after clot-selective coronary thrombolysis with Hybrid-B, a genetically engineered plasminogen activator with a prolonged biological half-life.

BACKGROUND: Despite the utility of tissue-type plasminogen activator (t-PA) in eliciting coronary thrombolysis clinically, early reocclusion remains a problem, occurring despite anticoagulation in 5-30% of patients with initially successful recanalization. This study evaluated the utility of Hybrid-B, a molecular variant of t-PA with a prolonged half-life in the circulation, in eliciting coronary thrombolysis and maintaining patency in the presence of a continuing thrombogenic stimulus. METHODS AND RESULTS: In intact, anesthetized dogs, either 18 mg Hybrid-B over 30 minutes (n = 15) or 50 mg t-PA (Activase) over 60 minutes (n = 8) was administered starting 60 minutes after left anterior descending coronary artery occlusion was induced with a thrombogenic copper coil. Time to lysis averaged 54 +/- 26 (means +/- SD) minutes and 64 +/- 34 minutes with Hybrid-B and t-PA, respectively (p = NS). When Hybrid-B was administered as a bolus (20 mg over 1 minute) to induce a high initial concentration in blood, time to lysis was shortened markedly and averaged 15 +/- 5 minutes. Dogs given Hybrid-B by either infusion or bolus exhibited prolonged time to reocclusion (337 +/- 192 minutes compared with 192 +/- 125 minutes in dogs given t-PA, p less than 0.03), reflecting maintenance of a subthrombolytic but persistently active concentration of activator in blood. Despite the persistence of Hybrid-B in blood, concentrations of fibrinogen and alpha 2-antiplasmin were not depleted markedly and remained at 77 +/- 25 and 56 +/- 24%, respectively, of control values. CONCLUSIONS: Thus, Hybrid-B, a novel variant of t-PA with unique pharmacokinetic properties, elicits prompt, sustained, and clot-selective coronary thrombolysis.

Deglycosylation increases the fibrinolytic activity of a deletion mutant of tissue-type plasminogen activator.

delta 2-89 t-PA is a deletion mutant lacking the finger (F) and epidermal growth factor (EGF) domains; thus, the fibrin interaction of this molecule must be mediated solely by the kringle region. In the present study, the influence of the oligosaccharide side-chains on the activity of delta 2-89 t-PA has been investigated. delta 2-89 t-PA was secreted in two forms, designated I and II, which presumably differ by the lack of one asparagine-linked oligosaccharide in the kringle 2 domain of form II. Forms I and II of delta 2-89 t-PA were purified; form II displayed higher fibrinolytic activity than form I. When form I was partially deglycosylated or treated to remove sialic acid, fibrinolytic activity was increased. Production of delta 2-89 t-PA in the presence of tunicamycin led to secretion of a glycan-free activator with higher activity. These findings suggest that certain oligosaccharide side-chains, particularly those containing sialic acid, can interfere with the interaction between the kringle region of t-PA and fibrin.

Alterations in the domain structure of tissue-type plasminogen activator change the nature of asparagine glycosylation.

The formation of N-linked oligosaccharides of eukaryotic glycoproteins starts with the attachment of a common precursor at the recognition site Asn-X-Ser/Thr. Subsequent processing, by yet unknown controlling factors, leads to the formation of three different glycans: the high mannose type, the complex type and the hybrid type. In order to gain insight into the processing mechanisms, we studied the glycan pattern of a panel of related molecules constructed by insertion, duplication or deletion of the domains encoded by the cDNA of a fibrinolytic glycoprotein, tissue-type plasminogen activator (t-PA). These variant molecules are identical in regard to the glycosylation sites originally situated in particular domains, but differ with respect to the sequential alignment of the domains. The variant and native t-PA genes were transfected into mouse C127 cells and their carbohydrate structures analyzed by the susceptibility to specific endoglycosidases and by reaction with sugar-specific lectins. We found that with one exception, all mutant activators lack the high mannose glycan found at asn 117 of native t-PA. The exception was a molecule that retains the original domain arrangement up to and through the glycosylation site at asn 117. These results demonstrate for the first time that structural alterations in the primary sequence distal to the actual glycosylation site can result in altered processing of N-linked oligosacharides.

Biological properties of hybrid plasminogen activators.

A number of hybrid plasminogen activator genes were constructed from the t-PA and u-PA cDNAs and expressed using a bovine papilloma virus vector and mouse C-127 cells. Hybrid A was constructed by replacing the finger (F) and EGF domains of t-PA with the EGF and Ku domains of u-PA, while hybrids B and C had an extra Ku inserted before or after the double kringle (K1-K2) region of t-PA respectively. While all the hybrids showed comparable enzymatic activities towards a small substrate (S-2288), they had different activities in binding to fibrin clots as well in the fibrin-dependent plasminogen activation, the order of activities being: t-PA greater than or equal to hybrid B greater than hybrid C greater than hybrid A. Carbohydrate analysis showed that while hybrid C, like rt-PA, had at least one high-mannose type sugar chain (probably at residue 117 in K1), the other hybrids had only complex-type carbohydrates suggesting that domain interaction in t-PA might influence glycan processing. Pharmacokinetic studies in dog showed that hybrid B had a significantly longer plasma half-life than rt-PA. Thrombolytic efficacies of hybrid B and rt-PA were compared in dog model using an artificially induced coronary thrombus. Complete thrombolysis was achieved with 18 mg and 50 mg dosages for hybrid B and rt-PA respectively. These data show the superior pharmacokinetic and thrombolytic properties of hybrid B compared to rt-PA.

Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis.

A 1.6 kb cDNA fragment encoding the mature part of the human tissue-type plasminogen activator (t-PA) was subcloned into a Bacillus subtilis dual plasmid expression system [Le Grice et al., Gene 55 (1987) 95-103]. Expression of the tPA gene in this vector was regulated by the inducible Escherichia coli lac elements, as well as a strong phage-T5-derived promoter and ribosome-binding site preceding the polylinker. The 5' end of the tPA gene corresponding to the N terminus of mature t-PA was fused in phase to the third codon present in the polylinker region of the expression vector, p602/22, to form p602-t-PA. B. subtilis containing p602-t-PA, when induced with isopropyl-beta-D-thiogalactopyranoside, produced large amounts of immunoreactive t-PA (approx. 20 micrograms/ml). As expected, t-PA was not secreted into the culture media, but was localized in intracellular inclusion bodies and was found to be enzymatically inactive. However, enzymatic activity could be regained following complete reduction followed by slow oxidation of the solubilized inclusion bodies. The recombinant t-PA (rt-PA) showed, after purification, a smaller molecular size than melanoma t-PA, probably due to lack of glycosylation in the Bacillus system. Like melanoma t-PA, rt-PA exhibited tremendous stimulation of plasminogen activation in the presence of fibrin. Our results illustrate that B. subtilis, when supplied with the proper transcriptional/translational regulatory elements, can be an effective system for expression of heterologous gene products.

Isolation of a human cDNA of urokinase and its expression in COS-1 cells.

The cDNA encoding human urokinase (UK) has been isolated from a cDNA library prepared from human normal fibroblast (WI38) cells, which had been stimulated by endothelial cell growth factor and heparin. This cDNA was sequenced and found to contain a few silent substitutions, thus encoding the same amino acids as deduced from the published genomic sequence of UK. After modification, the cDNA of UK was inserted into a transient expression vector and used to transfect COS-1 cells. The recombinant UK protein (rUK) in the serum-free medium of transfected COS-1 cells was characterized by biochemical and functional assays. These studies indicated that rUK from COS-1 cells is glycosylated, enzymatically active, and very similar to native single-chain plasminogen activator (scuPA). Therefore, such rUK can be a convenient source of scuPA for any further studies.

Expression and secretion of human atrial natriuretic alpha-factor in Bacillus subtilis using the subtilisin signal peptide.

Using the signal peptide of the Bacillus subtilis subtilisin gene (aprE) and a synthetic cDNA corresponding to the mature region of the human atrial natriuretic alpha-factor (hANF), we have constructed a secretion vector. B. subtilis cells, when transformed with this vector, secrete immunoreactive hANF peptides into the medium at about 500 micrograms/liter. The hANF is the first human gene product to be secreted from B. subtilis using this signal peptide. We have used promoters active during vegetative growth or sporulation and hosts deficient in several extracellular proteases but some proteolysis of the secretion products still occurs. In addition, both cell growth and sporulation are adversely affected by hANF production. Possible explanations for this observation are inefficient secretion of the atrial hormone or toxicity of the precursor or mature peptide.

Construction and expression of a hybrid plasminogen activator gene with sequences from non-protease region of tissue-type plasminogen activator (t-PA) and protease region of urokinase (u-PA).

There are two physiological plasminogen activators (PAs), tissue-type PA (t-PA) and urokinase (u-PA) which possess distinct immunological and biochemical characteristics. Using genetic engineering techniques a hybrid t:u-PA cDNA, comprised of amino acid (aa) sequences corresponding to the non-protease region (aa 1-261) of t-PA and the protease region (aa 132-411) of u-PA, was constructed. The t:u-PA gene after insertion into the SV40 expression vector was expressed in monkey Cos-1 cells. The 66-67 kDa t:u-PA was produced in an enzymatically active form. The fibrinolytic activity of the t:u-PA could be quenched by anti-urokinase as well as by anti-t-PA sera. Like urokinase, the t:u-PA showed a high intrinsic plasminogen activation. This activity, as in the case of t-PA, was stimulated by fibrin. The u-PA, on the other hand, stimulated plasminogen activation marginally in the presence of fibrin. Both the t:u-PA and t-PA showed binding affinity for fibrin clot. This study strongly suggests the autonomous nature of the structural domains in PA and also demonstrates the feasibility of shuffling these domains without loss of their functional activities.

Clearance of a novel recombinant tissue plasminogen activator in rabbits.

The pharmacokinetic properties of hPA(B), characterized by the insertion of a urokinase kringle coding region before the double kringle of tPA plus the complete tPA coding region, were investigated and compared to those of melanoma tPA (mtPA). Mean peak plasma concentrations at the end of infusion were 4.7 micrograms/ml for hPA(B) and 4.6 micrograms/ml for mtPA. The pharmacokinetics of both hPA(B) and mtPA showed a biexponential disappearance from plasma which is consistent with a two-compartment model of t 1/2 (lambda 1) = 2 minutes, t 1/2 (lambda 2) = 58 minutes for hPA(B), and t 1/2 (lambda 1) = 2.2 minutes, t 1/2 (lambda 2) = 61 minutes for mtPA. However, this very fast decaying lambda 1 phase of mtPA lasted five times longer than that of hPA(B) which resulted in very low concentrations of mtPA. Thus, hPA(B) exhibited larger AUC, slower clearance rate, and smaller volume of distribution (P less than 0.01) than those of mtPA. The fibrinolytic activity of hPA(B) in rabbit plasma as determined by zymography lasted up to 120 minutes after the end of infusion as compared to that of 2 minutes for mtPA. This indicates that mtPA, despite its t 1/2 (lambda 2) being similar to that of hPA(B), is no longer at physiologically meaningful concentrations at the start of the lambda 2 phase.

Disposition of a novel recombinant tissue plasminogen activator, delta 2-89 TPA, in mice.

The pharmacokinetic characteristics of delta 2-89 tPA, characterized by the deletion of the first 89 amino acids at the NH2-terminus of tPA, were evaluated and compared to those of recombinant tPA (rtPA). When they were administered intravenously to mice, a biexponential disposition curve was observed for both tPAs. The plasma half-lives of lambda 1 and lambda 2 phases of delta 2-89 tPA were 15 minutes and 180 minutes which are significantly higher than those of rtPA. A zymogram of mouse plasma taken at various time intervals showed that delta 2-89 tPA retained fibrinolytic activity up to 30 minutes, whereas rtPA could be detected only up to 5 minutes after injection. Autoradiography revealed that most of 125I-delta 2-89 tPA was associated with plasma protein complex.

Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties.

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.

Construction and expression of hybrid plasminogen activators prepared from tissue-type plasminogen activator and urokinase-type plasminogen activator genes.

Recent data from several studies have suggested that the non-protease domains in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) determine their biological specificities, including binding to fibrin clots and survival in the circulatory system (Van Zonneveld, A.-J., Veerman, H., and Pannekoek, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4670-4674; Rijken, D. C., and Emeis, J. J. (1986) Biochem. J. 238, 643-646). Structural manipulations (e.g. deletions, additions, or substitutions) in these domains can thus be utilized to maximize the desired biological effects. Using recombinant DNA technology, we constructed a number of hybrid molecules from the t-PA and u-PA genes. In hybrid A, the epidermal growth factor and finger domains of t-PA (residues 1-91) were replaced by the epidermal growth factor and kringle of u-PA (residues 1-131). In hybrids B and C, the u-PA kringle (residues 50-131) was inserted either before (residue 92) or after (residue 261) the double-kringle region of t-PA. All these hybrid PAs containing three kringles were expressed in mouse fibroblast cells (C-127). The hybrid proteins were synthesized in predominantly a single-chain form with molecular weights of 70,000-80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were enzymatically active as assayed by the fibrin-agar plate method. In vitro studies on the binding of hybrid PAs to fibrin showed that hybrid B, like t-PA, possesses affinity toward fibrin, while hybrid A shows lower binding. This suggests that the finger domain, which is not present in hybrid A, plays a role in conferring fibrin affinity to the hybrid PAs. The enzymatic activities of the hybrids were compared with that of recombinant t-PA (rt-PA) expressed in the same vector/host system and found to be similar in activity toward a chromogenic peptide substrate. In addition, plasminogen activation with all the hybrid-PAs, as with rt-PA, was stimulated by fibrin, with the order of activity being rt-PA greater than or equal to hybrid B greater than hybrid C greater than hybrid A. This study shows the feasibility of shuffling functional domain(s) of known specificity in plasminogen activators which may lead to the design of a superior thrombolytic agent.

Tissue plasminogen activator (tPA) as a reporter gene in transient gene expression.

Using the gene coding for tissue plasminogen activator (tPA) as a reporter gene, a transient gene expression system has been established. Vectors containing the full-length cDNA of tPA with its signal sequences were introduced into mammalian recipient cells by a modified gene transfer procedure. Thirty hours after transfection, the secreted tPA was found in serum-free medium and measured by a fibrin-agarose plate assay (FAPA). In this assay, tPA converts plasminogen into plasmin which then degrades high-Mr fibrin to produce cleared zones. The sizes of these zones correspond to quantities of tPA. The combination of transient tPA expression system and the FAPA provides a quick, sensitive, quantitative and non-destructive method to examine the strength of eukaryotic regulatory elements in tissue-culture cells.

Site-specific DNA splicing: a general procedure for the creation of a restriction site at a predetermined position in a DNA sequence.

A method is described for creating any of a wide array of restriction sites at a predetermined position in a known DNA sequence. The method utilizes the exonuclease activity of BAL 31 and a specially designed bifunctional oligodeoxynucleotide linker. The desired restriction site is generated when the linker is ligated to those BAL 31-digested DNA fragments which end with the target sequence. The proper ligation product is then identified by a highly specific hybridization procedure. The method is versatile and specific and is especially useful in the isolation of functional elements of a gene.