Three domains in the simian virus 40 core origin orchestrate the binding, melting, and DNA helicase activities of T antigen.

The simian virus 40 (SV40) core origin of replication consists of three functional domains. The sequence 5'-CACTACTTCTGGAATAG-3' with an imperfect inverted repeat (underlined), a palindrome with four 5'-GAGGC-3' pentanucleotide repeats, and a 17-base-pair A + T-rich segment. We have been able to assign primary functions to each domain. Remarkably, SV40 large T antigen melted the inverted repeat domain in the complete absence of other origin sequences. Presumably, this protein-DNA interaction initiates a replication bubble that leads to daughter strand DNA synthesis. The pentanucleotide domain alone docked and arranged T antigen at the origin. The A + T-rich domain had no independent function, but, in the presence of the other two domains, allowed bound T antigen to extend the replication bubble. Thus, three domains of the origin coordinate the binding, melting, and DNA helicase activities of T antigen in an ordered sequence of events to initiate DNA replication.     

Induction of Neuroendocrine Differentiation in Prostate Cancer Cells by Dovitinib (TKI-258) and its Therapeutic Implications

Prostate cancer (PCa) remains the second-leading cause of cancer-related deaths in American men with an estimated mortality of more than 26,000 in 2016 alone. Aggressive and metastatic tumors are treated with androgen deprivation therapies (ADT); however, the tumors acquire resistance and develop into lethal castration resistant prostate cancer (CRPC). With the advent of better therapeutics, the incidences of a more aggressive neuroendocrine prostate cancer (NEPC) variant continue to emerge. Although de novo occurrences of NEPC are rare, more than 25% of the therapy-resistant patients on highly potent new-generation anti-androgen therapies end up with NEPC. This, along with previous observations of an increase in the number of such NE cells in aggressive tumors, has been suggested as a mechanism of resistance development during prostate cancer progression. Dovitinib (TKI-258/CHIR-258) is a pan receptor tyrosine kinase (RTK) inhibitor that targets VEGFR, FGFR, PDGFR, and KIT. It has shown efficacy in mouse-model of PCa bone metastasis, and is presently in clinical trials for several cancers. We observed that both androgen receptor (AR) positive and AR-negative PCa cells differentiate into a NE phenotype upon treatment with Dovitinib. The NE differentiation was also observed when mice harboring PC3-xenografted tumors were systemically treated with Dovitinib. The mechanistic underpinnings of this differentiation are unclear, but seem to be supported through MAPK-, PI3K-, and Wnt-signaling pathways. Further elucidation of the differentiation process will enable the identification of alternative salvage or combination therapies to overcome the potential resistance development.     

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.     

Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus.

The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c-src. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c-src C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity.     

Effects of thyroxine-driven precocious metamorphosis on maturation of adult-type allograft rejection responses in early thyroidectomized frogs

In contrast to the adult pattern of allograft reactivity, larval South African clawed frogs (Xenopus) either become tolerant of adult major histocompatibility complex (MHC) disparate skin grafts or reject them slowly. Larvae fail to reject grafts that are incompatible at minor histocompatibility (H) loci (MHC-identical); rather, they become immunologically tolerant. We report here that early thyroidectomized (thyroidx) larvae, forced to metamorphose precociously on a regimen of thyroxine (T4) treatment, showed larva-like responses in that their ability to reject skin allografts that differed by minor H loci or one MHC haplotype was significantly impaired relative to age-matched intact postmetamorphic controls. During the normal ontogeny of Xenopus, lymphocyte numbers increase in larval life, decrease during metamorphosis, and increase to adult levels at 6-12 months of age. Thyroidectomy and a low-dose regimen of thyroxine treatment limited the number of lymphocytes that developed in larval organs and very dramatically reduced those populations below control levels during metamorphosis. Because these changes were stage-dependent rather than age-dependent, the second wave of lymphopoiesis in thyroidx frogs treated with low T4 may occur out of synchrony with the expression of Class I MHC antigens. Impaired allograft rejection, therefore, may reflect the absence of a population of cells that can effectively recognize minor H alloantigens and/or self and allo Class I antigens.     

National and international regulations governing transportation and supply of primate animals

Import and export of primates is controlled by the Endangered Species Act and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Permits are required for all international transactions, and certain primates may not be traded at all for commercial permits.     

Impaired Response Inhibition in the Rat 5 Choice Continuous Performance Task during Protracted Abstinence from Chronic Alcohol Consumption

Impaired cognitive processing is a hallmark of addiction. In particular, deficits in inhibitory control can propel continued drug use despite adverse consequences. Clinical evidence shows that detoxified alcoholics exhibit poor inhibitory control in the Continuous Performance Task (CPT) and related tests of motor impulsivity. Animal models may provide important insight into the neural mechanisms underlying this consequence of chronic alcohol exposure though pre-clinical investigations of behavioral inhibition during alcohol abstinence are sparse. The present study employed the rat 5 Choice-Continuous Performance Task (5C-CPT), a novel pre-clinical variant of the CPT, to evaluate attentional capacity and impulse control over the course of protracted abstinence from chronic intermittent alcohol consumption. In tests conducted with familiar 5C-CPT conditions EtOH-exposed rats exhibited impaired attentional capacity during the first hours of abstinence and impaired behavioral restraint (increased false alarms) during the first 5d of abstinence that dissipated thereafter. Subsequent tests employing visual distractors that increase the cognitive load of the task revealed significant increases in impulsive action (premature responses) at 3 and 5 weeks of abstinence, and the emergence of impaired behavioral restraint (increased false alarms) at 7 weeks of abstinence. Collectively, these findings demonstrate the emergence of increased impulsive action in alcohol-dependent rats during protracted alcohol abstinence and suggest the 5C-CPT with visual distractors may provide a viable behavioral platform for characterizing the neurobiological substrates underlying impaired behavioral inhibition resulting from chronic intermittent alcohol exposure.     

Collaborative Filtering for Brain-Computer Interaction Using Transfer Learning and Active Class Selection

Brain-computer interaction (BCI) and physiological computing are terms that refer to using processed neural or physiological signals to influence human interaction with computers, environment, and each other. A major challenge in developing these systems arises from the large individual differences typically seen in the neural/physiological responses. As a result, many researchers use individually-trained recognition algorithms to process this data. In order to minimize time, cost, and barriers to use, there is a need to minimize the amount of individual training data required, or equivalently, to increase the recognition accuracy without increasing the number of user-specific training samples. One promising method for achieving this is collaborative filtering, which combines training data from the individual subject with additional training data from other, similar subjects. This paper describes a successful application of a collaborative filtering approach intended for a BCI system. This approach is based on transfer learning (TL), active class selection (ACS), and a mean squared difference user-similarity heuristic. The resulting BCI system uses neural and physiological signals for automatic task difficulty recognition. TL improves the learning performance by combining a small number of user-specific training samples with a large number of auxiliary training samples from other similar subjects. ACS optimally selects the classes to generate user-specific training samples. Experimental results on 18 subjects, using both nearest neighbors and support vector machine classifiers, demonstrate that the proposed approach can significantly reduce the number of user-specific training data samples. This collaborative filtering approach will also be generalizable to handling individual differences in many other applications that involve human neural or physiological data, such as affective computing.