Long-lasting adenovirus transgene expression in mice through neonatal intrathymic tolerance induction without the use of immunosuppression.

The major barrier to the clinical application of adenovirus gene therapy for diseases that require stable transgene expression is the immunogenicity of recombinant adenovirus, which ordinarily limits the duration of its effects to a period of about 2 weeks. We postulated that tolerance to adenovirus could be induced and transgene expression could be prolonged if T lymphocytes underwent thymic selection in the presence of adenovirus antigens. Mice were inoculated in the thymus with a recombinant adenovirus containing the lacZ marker gene during the neonatal period at a time before T-cell maturation had occurred. When the virus was administered intravenously to these mice in adulthood, they were found to have an impaired adenovirus-specific cytotoxic T-lymphocyte response which allowed prolonged hepatic lacZ expression, for up to 260 days. The ability to achieve unresponsiveness to a recombinant adenovirus after inoculation of the thymus in neonates extends the paradigm of intrathymic tolerance induction. Furthermore, this model will enable the study of stable adenovirus transgene expression in vivo without the use of immunosuppression and ultimately may have clinical utility.     

L-Asparagainases from Citrobacter freundii.

Three enzymes which catalyze the hydrolysis of L-asparagine have been identified in extracts of Citrobacter freundii. One of these (asparaginase-glutaminase (EC 3.5.1.1) also shows substantial glutaminase activity. This enzyme is extremely labile, is sensitive to inactivation by p-chloromercuribenzoate, and is not protected by dithiothreitol. A second enzyme (asparaginase B) is also sensitive to mercurials but is protected from inactivation by dithiothreitol. This enzyme has a relatively low affinity for L-asparagine (Km = 1.7-10(-3) M). The third enzyme (asparaginase A) is insensitive to inactivation by mercurials, is stable upon long term storage and has a relatively high affinity for L-asparagine (Km = 2.9-10(-5) M). This enzyme has been purified to homogeneity and has a molecular weight of approx. 140 000; the subunit weight being approx. 33 000. The C. freundii asparaginase A produced significant increases in the survival time of C3H/HE mice carrying the 6C3HED lymphoma tumor.     

Serotonin sensitive adenylate cyclase activity in monkey anterior limbic cortex: antagonism by molindone and other antipsychotic drugs.

Serotonin (5-HT) sensitive adenylate cyclase in monkey anterior limbic cortex homogenates was further characterized and the effects of antipsychotic drugs and 5-HT anatagonists investigated. Differences in time course for stimulation by agonists and in responsiveness to receptor anatagonists of 5-HT-and dopamine (DA)-stimulated activities, were observed. Also there was an additivity of 5-HT and DA at maximally effective concentrations. Classical 5-HT antagonists blocked the 5-HT sensitive adenylate cyclase with a rank order of potency: methiothepin > cyproheptadine > methysergide. These 5-HT antagonists also effectively inhibited DA sensitive adenylate cyclase. Most antipsychotic drugs tested antagonized 5-HT stimulated activity although these drugs exhibited greater efficacies in blocking DA stimulated activity. Exceptions were molindone which failed to antagonize DA sensitive adenylate cyclase but effectively blocked 5-HT sensitive cyclase and pipamperone which was inactive in both cyclase systems. Haloperidol was a more selective antagonist of the DA sensitive adenylate cyclase than were the other antipsychotic drugs tested.     

3H-Spiroperidol binding and dopamine-stimulated adenylate cyclase: evidence for multiple classes of receptors in primate brain regions.

Studies of displacement by agonist and antagonist drugs of ³H-spiroperidol binding in brain regions of $\text{Cebus}$ and rhesus monkeys revealed one type of receptor in caudate nucleus and a second type of receptor in both frontal and anterior limbic cortex. Compared with caudate, the cortical regions were more sensitive to clozapine and loxapine, equally sensitive to fluphenazine and relatively less sensitive to haloperidol. Also, the cortical regions were insensitive to molindone. Parallel studies using the dopamine-stimulated adenylate cyclase have demonstrated three types of receptors, one in caudate, a second in frontal cortex, and a third in anterior limbic cortex. In each region studied, relative sensitivities to drug using these two methods differed, suggesting that in each of these regions only a relatively small portion of ³H-spiroperidol receptors are coupled to adenylate cyclase.     

Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase.

Epidermal growth factor stimulates the activity of several cytosolic serine/threonine protein kinases in quiescent Swiss 3T3 cells. Two of these, which use myelin basic protein (MBP) as substrate, act as kinase kinases in that they are able to activate a separate peptide kinase activity in vitro by a mechanism involving protein phosphorylation. In this study, we have identified two activities from extracts of epidermal growth factor-treated cells that stimulate an ATP-dependent activation of both of the MBP kinases, derived in their inactive precursor forms from extracts of untreated cells. The resulting MBP kinase activities are stable to further purification and can be inactivated with either tyrosine or serine/threonine protein phosphatases and then reactivated to their original levels of activity. Thus, we propose that the in vitro activation involves protein phosphorylation, stimulated by the action of novel MBP kinase activating factors that represent intermediate components in a growth factor-stimulated kinase cascade.     

Expression of glucoamylase gene usingSUC2 promoter inSaccharomyces cerevisiae

Summary The cloning of glucoamylase geneSTA using theSUC2 promoter intoSaccharomyces cerevisiae was performed. The signal sequence ofSTA gene was used for the secretion of glucoamylase protein. The plasmid constructed in this way was named YEpSUCSTA and its expression was identified. The expression of YEpSUCSTA was repressed in the presence of glucose in growth medium, but derepressed when glucose became depleted. YEpSUCSTA showed the similar efficiency of glucoamylase secretion as YEpSTA-F which has the entireSTA gene. Glucoamylase activity in starch-glucose medium was largely increased because cell mass and plasmid stability were high in biosynthesis phase compared to extracellular glucoamylase activities in media which starch or glucose was the only carbon source.