Targeting of tissue plasminogen activator into the regulated secretory pathway of neuroendocrine cells

Plasma levels of tissue plasminogen activator (tPA) increase rapidly in response to specific vasoactive agents, trauma, and neural stimulation. This response has been attributed to acute release of tPA from stored pools within the vascular endothelium and from catecholamine storage vesicles of chromaffin cells. We have tested directly whether tPA can be sorted into the regulated secretory pathway using the murine pituitary-derived neuroendocrine cell line AtT-20 transfected with tPA cDNA. Clones of AtT-20 cells expressing tPA were isolated, and targeting of tPA into the regulated secretory pathway was demonstrated by (1) stimulation of tPA secretion with 8-bromo-cAMP, the secretagogue which promotes the release of dense granule contents; (2) colocalization with ACTH, an endogenous protein that is stored in dense core granules; and (3) retention of newly synthesized tPA in the cell for prolonged periods of time. Laser scanning confocal microscopy analysis of cells immunostained with antibodies to tPA and ACTH showed colocalization at the tips of the neuritic processes under the cytoplasmic membrane, a region where dense granules are known to migrate after maturation. Treatment of the cells with 5 mM 8-bromo-cAMP for 30 min resulted in a 2.41+/-0.36-fold increase in tPA secretion. Both the magnitude of the stimulatory effect and the fraction of the intracellular tPA released were the same regardless of the tPA expression level in the various clones. Pulse-chase experiments showed that a portion of newly synthesized tPA is retained in the cell for at least 4 h and is released into the culture medium in response to 8-bromo-cAMP. These studies indicate that tPA, under the appropriate conditions, can be targeted into the regulated secretory pathway and can be stored for later release by cellular stimuli.     

Role of microwave heating strategies in enhancing the progress of a first‐order endothermic reaction

A detailed analysis has been carried out by solving coupled energy and mass balance equations along with Helmholtz equation of microwave propagation to investigate the role of microwave heating strategies in enhancing the progress of a first‐order endothermic reaction in “thin,” “intermediate,” and “thick” regimes of microwave heating. It has been shown that heating strategy plays a critical role in maximizing the efficiency of microwave heated reactors, where progress of reactor can be greatly accelerated at a given input power by either configuring microwave sources at both sides of the reactor or preferentially focusing them on one side. A series of master curves have been provided showing percentage saving of reaction time in various configurations of microwave heated reactors compared to their conventional counterparts, which can be efficiently used to select appropriate heating strategy for different dimension and dielectric properties of the reacting medium. © 2012 American Institute of Chemical Engineers AIChE J, 59: 656–670, 2013     

Effects of marital status on the risk of mortality in poor and non-poor neighborhoods

PURPOSE: The purpose of this paper is to consider whether the mortality risks associated with marital status are conditioned by the socioeconomic quality of neighborhoods. METHODS: The analysis is based on the first National Health and Nutrition Examination Survey 1971-1974 (NHANES I), and the 1987 NHANES I Epidemiologic Followup Survey (NHEFS). Cox proportional hazards regressions were used to assess whether the effect of marital status on the risk of all-cause and cause-specific mortality is altered by local area poverty. Analyses are stratified by age, sex, and urbanicity. RESULTS: The interaction between neighborhood poverty and marital status is suggested for non-elderly men, particularly for cancer mortality and for men in urban areas. Interaction effects are evident among older women residing in urban areas. CONCLUSIONS: The absence of a spouse elevates the risk of mortality but this risk is moderately higher in impoverished neighborhoods, notably in urbanized areas, for non-elderly men and elderly women. Future studies with larger samples of non-married persons where marital status changes are incorporated are needed to improve our understanding of the joint mortality effects of local area poverty and marital status.     

Doppler quantification of echo-contrast injections in vivo

It is difficult to quantify myocardial perfusion using contrast echocardiography because the echogenicity of injected contrast is unknown. We propose that a measurement of Doppler amplitude from blood in a systemic artery during the passage of contrast could define the needed input function. Time-amplitude curves from pulsed Doppler cuffs on coronary and carotid arteries of 7 dogs were analyzed during aortic root and left atrial injections of Albunex. We found in individual animals that the areas under the Doppler time-amplitude curves were correlated to the amount of Albunex injected (R = 0.87-0.99), inversely correlated to cardiac output (R = 0.83), and uncorrelated to coronary flow (R = 0.18). Due to better mixing, the coronary and carotid response areas correlated better for left atrial injections (R = 0.96) than for aortic root injections (R = 0.56). We conclude that Doppler amplitude detection can be used to quantify the passage of echo-contrast agents, that the measurements comply with indicator-dilution principles, and that systemic measurements in the carotid artery could be used to predict the coronary input function for injection sites with good systemic mixing.     

Identification and structural influence of a differentially modified N-terminal methionine in human S100b

The calcium-binding protein S100b is a homodimer comprised of two identical 91-residue beta-subunits. Recombinant S100b is a heterogeneous protein, although the basis of this heterogeneity has not been established. We have used mass spectrometry and NMR spectroscopy to determine that heterogeneity in S100b arises from a mixture of formyl-S100b and desformyl-S100b when expressed in Escherichia coli. Reversed-phase HPLC purification of these two forms of S100b has allowed the differences in N-terminal composition to be used as a probe for tertiary contacts in the protein. The presence or absence of the N-terminal formyl group affected the chemical shifts of sequence neighboring residues and those in the linker of the protein (residues 40-43), indicating that these two regions are close in space.     

Functional MRI reveals left amygdala activation during emotion

The potential of functional magnetic resonance imaging (fMRI) for experimental studies of the brain and behavior considerable given its superior time and spatial resolution, but few studies have attempted to validate them against established methods for measuring cerebral activation. In a previous study absolute regional cerebral blood flow was measured in 16 healthy individuals using quantitative H215O-PET during standardized happy and sad mood induction and during two non-emotional control conditions. During sad mood, blood flow increased in the left amygdala and these changes correlated with shifts towards a negative affect. In the present study blood oxygenation level dependent (BOLD) changes were measured with fMRI during the same experimentally controlled mood states and control tasks. Twelve right-handed normal subjects were examined with a T2*-weighted FLASH sequence. A significant increase in signal intensity was found during sad as well as happy mood induction in the left amygdala. This converging evidence supports the potential of fMRI for advancing the understanding of neural substrates for emotional experience in humans.     

Renal styphlodoriasis in a boa constrictor

Renal styphlodoriasis was diagnosed in a 2 1/2-year-old male boa constrictor (Constrictor constrictor). The snake had been anorexic for 6 months prior to its death. Necropsy revealed numerous hard, white foci of mineralization in the kidneys. Histologic examination revealed distorted renal tubules containing cross sections of trematodes or mineralized debris, tubular epithelial hyperplasia, and chronic interstitial nephritis. Several adult trematodes were recovered and identified as Styphlodora horrida.     

Functional and DNA sequence divergence of the CYP71 gene family in higher plants

During ripening of avocado (Persea americana), the CYP71A1 mRNA and protein accumulate to relatively high levels. Although the CYP71A1 gene was the first plant P450 to be cloned and sequenced, the functional role of this P450 remains obscure. Substrate studies have shown that CYP71A1 will metabolize various monoterpenes (nerol and geraniol), although these have not been detected in ripening fruit. Using DNA from a conserved domain of the CYP71A1 gene, we have explored the scope of the CYP71 (or related) gene family in avocado using low stringency DNA hybridization. This analysis suggests that there are approximately 10-12 genes in the CYP71 family. An alternative approach using PCR gave essentially identical results.