Cellular receptors for sex steroids in human pituitary adenomas

Cellular receptors for sex steroids (SSRs) were studied in an unselected series of 55 human pituitary tumors. Cytosolic receptors for estrogen (ERcs) and progesterone (PgRcs) were determined in all cases and cytosolic androgen receptors (ARcs) in 47 cases. Nuclear receptors (ERns, PgRns, ARns) were also studied in 33 cases. ERs and PgRs were determined by an ELISA and ARs by [3H]methyltrienolone binding. Where both cytosolic and nuclear receptors were studied (n = 33), ERs, PgRs and ARs were found in at least one subcellular fraction in 66.7, 60.6 and 81.8% of cases respectively, ERs and ARs being mainly recovered from the cytosol and PgRs from the nucleus. No linear correlation was found between pre-operative plasma steroid hormones and their specific cellular receptors. Nonetheless, the differential expression of SSRs according to sex and gonadal status at the time of surgery strongly supports their regulation by the steroid environment in vivo: PgRcs were more frequent in tumors found in women (41.4 vs 15.4%, P < 0.05), whereas a high expression of ERcs and ARcs (> 15 fmol/mg protein) was more common in tumors found in men (34.5 vs 10.3%, P < 0.05 and 54.5 vs 24.0% respectively). PgRs were positively correlated with ERns, indicating the possibility of estrogen priming of their expression, and negatively correlated with ARs in nuclear fractions. SSRs appeared to be widely distributed among pituitary tumors, although, compared with other hormone-secreting groups, prolactinomas displayed a higher ERc expression (34.8 +/- 11.3 vs 4.8 +/- 5.1 fmol/mg protein, P = 0.007) and gonadotroph cell adenomas lower ARc values (1.3 +/- 0.8 vs 38.2 +/- 10.6 fmol/mg protein, P = 0.048). Microadenomas were characterized by a higher PgR expression than macroadenomas, whereas hemorrhagic (macro)adenomas were characterized by a high ER expression (> 90%). The present results indicate that most pituitary tumors are targets for sex steroids, SSR expression being partially triggered by the steroid environment itself. Possible physiopathological and therapeutic implications of these findings are discussed.     

Assessment of the biodegradation potential of psychrotrophic microorganisms

These experiments were designed to establish an animal model of acute carbon monoxide (CO) poisoning in awake habituated rats. On the day before exposure, under a brief anesthesia, a Levine preparation (unilateral common carotid artery occlusion) was performed on group 1 (n = 8) and 2 (n = 28), but not on group 3 rats (n = 8). Group 1 rats were exposed to air as control. Groups 2 and 3 rats were exposed to 0.27% CO in air for 60 min [carboxyhemoglobin (HbCO) = 70%] followed by a 2-day recovery in air. The Levine preparation per se did not induce any detectable physiologic effects on group 1 rats. Identical cardiovascular and metabolic responses to CO occurred in groups 2 and 3. After the CO exposure, all group 3 rats lived for 2 days with normal neurologic index (NI). In group 2 (n = 25 post-CO), 84% of the rats showed increased NI and edema of the ipsilateral cerebral hemisphere, and 76% of the rats died 8.7 +/- 1.7 h after the CO exposure. NI correlated with the brain edema (rs = 0.748, P < 0.001) and inversely correlated with the survival time after the CO exposure (rs = -0.777, P < 0.001). We therefore may conclude that exposure of the Levine-prepared rats to 0.27% CO in air for 60 min will provide a valuable model for testing of different treatments for CO poisoning.     

Breathing of awake goats during prolonged dysfunction of caudal M ventrolateral medullary neurons

Recent scientific studies have demonstrated the efficacy of various forms of immunotherapy for the treatment of allergic diseases. Traditional subcutaneous immunotherapy, sublingual, oral, and intranasal immunotherapy have been shown to significantly reduce symptoms and favorably modulate the immune response. Outcome studies that use patient response data from standardized surveys represent the next challenge to all practicing allergists.     

Sorting human beta-cells consequent to targeted expression of green fluorescent protein

Pancreatic islets of Langerhans are composed of four major endocrine cell types with a smaller number of nonendocrine cells. To study the molecular constituents and function of just one subpopulation of islet cells, it is necessary to sort them from the other cell types. While rat beta-cells can be sorted by autofluorescence-activated flow cytometry, this has not proved possible on a routine and reproducible basis for human beta-cells. In the present study, we have selectively labeled human beta-cells with green fluorescent protein (GFP), allowing for their sorting by flow cytometry. Human islet cells were infected with replication-defective (attenuated) recombinant adenovirus expressing GFP driven by the rat insulin I promoter (Ad-RIP-GFP) for targeted expression in beta-cells, or beta-galactosidase driven by the promiscuous cytomegalovirus (CMV) promoter (Ad-CMV-beta-gal) as control. Whereas the majority of islet cells can be infected by adenovirus, as shown by control infection with Ad-CMV-beta-gal, increased fluorescence after infection with Ad-RIP-GFP was limited to insulin-containing beta-cells. Infection of islet cells with Ad-RIP-GFP resulted reproducibly in the appearance of a population of intensely fluorescent cells, when analyzed by flow cytometry. These cells were sorted using a fluorescence-activated cell sorter (FACS) and shown by immunofluorescence to consist of >95% beta-cells. The targeted expression of GFP thus allows for preparation of human beta-cells purified close to homogeneity. This method should be readily applicable in any laboratory with FACS capability.     

Intrathoracic tracheal avulsion and pseudodiverticulum following pneumomediastinum in a cat

A case of tracheal avulsion with pneumomediastinum and pseudodiverticulum formation in a cat is described. Radiographs taken at initial presentation showed a pneumomediastinum with fractures of the right first to sixth ribs. Conservative treatment resolved the pneumomediastinum but following initial improvement the cat developed dyspnoea. Radiography and image intensification at this time showed a tracheal diverticulum at the level of the fourth rib. Surgical resection of the diverticulum and stenotic tracheal ends was performed via a right fourth intercostal lateral thoracotomy. The airway was maintained throughout the procedure using a bridging endotracheal tube. The cat made an unremarkable recovery following surgery.     

Phosphoinositide 3-kinase regulates phospholipase Cgamma-mediated calcium signaling

It has been demonstrated that the lipid products of the phosphoinositide 3-kinase (PI3K) can associate with the Src homology 2 (SH2) domains of specific signaling molecules and modify their actions. In the current experiments, phosphatidylinositol 3,4, 5-trisphosphate (PtdIns-3,4,5-P3) was found to bind to the C-terminal SH2 domain of phospholipase Cgamma (PLCgamma) with an apparent Kd of 2.4 microM and to displace the C-terminal SH2 domain from the activated platelet-derived growth factor receptor (PDGFR). To investigate the in vivo relevance of this observation, intracellular inositol trisphosphate (IP3) generation and calcium release were examined in HepG2 cells expressing a series of PDGFR mutants that activate PLCgamma with or without receptor association with PI3K. Coactivation of PLCgamma and PI3K resulted in an approximately 40% increase in both intracellular IP3 generation and intracellular calcium release as compared with selective activation of PLCgamma. Similarly, the addition of wortmannin or LY294002 to cells expressing the wild-type PDGFR inhibited the release of intracellular calcium. Thus, generation of PtdIns-3,4,5-P3 by receptor-associated PI3K causes an increase in IP3 production and intracellular calcium release, potentially via enhanced PtdIns-4, 5-P2 substrate availability due to PtdIns-3,4,5-P3-mediated recruitment of PLCgamma to the lipid bilayer.     

Tumor regression in mice following vaccination with human papillomavirus E7 recombinant protein in PROVAX

Induction of CD8+ cytotoxic T lymphocytes (CTLs) specific for human papillomavirus (HPV) antigens provides an attractive strategy for immunotherapy of HPV-related cancers in humans. In this study, we investigated the potential of utilizing soluble E7 protein of HPV 16 in an adjuvant formulation, PROVAX as a vaccine against a progressively growing E7 transfected K1735-X21 (H-2k) metastatic melanoma cells (HOPE2) in a mouse model. Vaccination of HOPE2 tumor bearing mice (C3H) with E7 protein in PROVAX resulted in significant inhibition of tumor growth, compared to mice vaccinated with E7 in Alum or saline. In vivo depletion of CD8+ or CD4+ cells indicated that CD8+ cells are the major effector cells in mediating the anti-tumor activity in this model. Furthermore, E7-specific CTL activity in vitro was detected in tumor bearing mice vaccinated with E7-PROVAX. Our studies suggest that recombinant HPV antigens in combination with PROVAX could serve as an effective subunit vaccine to stimulate tumor specific CD8+ T cell mediated immunity against HPV-related cancers.     

The role of prefrontal cortex in working memory: examining the contents of consciousness

Working memory enables us to hold in our 'mind's eye' the contents of our conscious awareness, even in the absence of sensory input, by maintaining an active representation of information for a brief period of time. In this review we consider the functional organization of the prefrontal cortex and its role in this cognitive process. First, we present evidence from brain-imaging studies that prefrontal cortex shows sustained activity during the delay period of visual working memory tasks, indicating that this cortex maintains on-line representations of stimuli after they are removed from view. We then present evidence for domain specificity within frontal cortex based on the type of information, with object working memory mediated by more ventral frontal regions and spatial working memory mediated by more dorsal frontal regions. We also propose that a second dimension for domain specificity within prefrontal cortex might exist for object working memory on the basis of the type of representation, with analytic representations maintained preferentially in the left hemisphere and image-based representations maintained preferentially in the right hemisphere. Furthermore, we discuss the possibility that there are prefrontal areas brought into play during the monitoring and manipulation of information in working memory in addition to those engaged during the maintenance of this information. Finally, we consider the relationship of prefrontal areas important for working memory, both to posterior visual processing areas and to prefrontal areas associated with long-term memory.     

The development of perceptual completion abilities: infants' perception of stationary, partially occluded objects

We explored infants' ability to perceive stationary, partially occluded objects as connected units (Experiments 1 and 2) with specific appearances (Experiment 3). In each experiment, the infants saw 2 test events involving what appeared to adults to be a tall rectangular object whose middle portion was hidden behind a narrow screen. During the test events, the screen alternately uncovered and covered the object. In Experiments 1 and 2, removal of the screen revealed either a single, connected rectangle (complete object event) or an interrupted rectangle with a gap where the screen had been (broken object event). In Experiment 3, removal of the screen revealed either a rectangle (rectangle event) or a cross-shaped object (cross-shape event). The pattern of infants' looking times at these events suggest that they perceive the unity of the partially occluded object by 6.5 months of age but did not perceive the form of the hidden part of the object until 8 months. The results of baseline control conditions support this interpretation.