Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1

The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines.     

Adolescent Changes in Dopamine D1 Receptor Expression in Orbitofrontal Cortex and Piriform Cortex Accompany an Associative Learning Deficit

The orbitofrontal cortex (OFC) and piriform cortex are involved in encoding the predictive value of olfactory stimuli in rats, and neural responses to olfactory stimuli in these areas change as associations are learned. This experience-dependent plasticity mirrors task-related changes previously observed in mesocortical dopamine neurons, which have been implicated in learning the predictive value of cues. Although forms of associative learning can be found at all ages, cortical dopamine projections do not mature until after postnatal day 35 in the rat. We hypothesized that these changes in dopamine circuitry during the juvenile and adolescent periods would result in age-dependent differences in learning the predictive value of environmental cues. Using an odor-guided associative learning task, we found that adolescent rats learn the association between an odor and a palatable reward significantly more slowly than either juvenile or adult rats. Further, adolescent rats displayed greater distractibility during the task than either juvenile or adult rats. Using real-time quantitative PCR and immunohistochemical methods, we observed that the behavioral deficit in adolescence coincides with a significant increase in D1 dopamine receptor expression compared to juvenile rats in both the OFC and piriform cortex. Further, we found that both the slower learning and increased distractibility exhibited in adolescence could be alleviated by experience with the association task as a juvenile, or by an acute administration of a low dose of either the dopamine D1 receptor agonist SKF-38393 or the D2 receptor antagonist eticlopride. These results suggest that dopaminergic modulation of cortical function may be important for learning the predictive value of environmental stimuli, and that developmental changes in cortical dopaminergic circuitry may underlie age-related differences in associative learning.     

Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro

The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi followed by TCM-199 + 10% ECS from 72 to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.     

Live imaging early immune cell ontogeny and function in zebrafish Danio rerio

The success of a robust vertebrate inflammatory response is in part because of the migratory potential of its haematopoietic components. Once these cells converge at an inflammatory site, they interact with each other as well as non‐immune tissues and infectious agents to help manage both the scale and the duration of any ensuing response. Exactly how these blood cells, that constitute the innate and adaptive immune systems, contribute to such immune responses remain largely unknown. Traditionally, assessing these contributions relied upon histological analysis of fixed tissue sections complemented with in vitro dynamic data. Although informative, translating results from these studies into the multicellular whole‐animal setting remain difficult. Recently, non‐invasive live imaging of the immune system in animal models is providing significant insights into how immune cells function within their intact natural environment. Although the majority of these studies have been conducted within mice, another vertebrate, the zebrafish Danio rerio is being recognized as an ideal platform for non‐invasive live imaging applications. The optical transparency, rapid development, genetic tractability and highly conserved innate and adaptive immune systems of this well‐established developmental model have been exploited in a number of recent studies evaluating the immunocompetence of fluorescently tagged blood cells. In addition, similar live imaging studies are helping to dissect the ontogeny of blood‐cell development by tracking various haematopoietic precursor cells to assess their contribution to different blood lineages. This review will examine some recent advances that have helped D. rerio emerge as a live imaging platform as well as its potential to offer valuable insights into the genetics behind diseases associated with immune cell dysfunction.