On the mechanism by which dopamine inhibits prolactin release in the anterior pituitary

An $\text{in}$$\text{vitro}$ perfusion system was used to assess the effects of chloride channel blockers, dopamine (DA) receptor agonists and antagonists, and GABA receptor agonists and antagonists on prolactin release from the mouse anterior pituitary. Dopamine and muscimol inhibited prolactin release ($\text{IC}{}_{50}{}^1\text{= 6 × 10}{}^{\mathrm{?8}}\text{M and}\text{10}{}^{\mathrm{?5}}\text{M}$ respectively). The GABA receptor antagonist bicuculline blocked the inhibition of prolactin release by muscimol but not dopamine. The dopamine receptor antagonist chlorpromazine blocked the dopamine- but not muscimol-induced inhibition of prolactin release. Haloperidol, however, reversed both the muscimol and dopamine induced inhibition of prolactin release. Furthermore, the chloride channel blocker picrotoxinin blocked the inhibition of prolactin release elicited by both dopamine and muscimol. These later results suggest that the anterior pituitary dopamine receptor which mediates the inhibition of prolactin release may be coupled to a picrotoxinin sensitive chloride ionophore and that haloperidol may affect the function of both DA and GABA receptors in the anterior pituitary.     

Environmental Particulate (PM2.5) Augments Stiffness-Induced Alveolar Epithelial Cell Mechanoactivation of Transforming Growth Factor Beta

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor - beta (TGFβ) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.     

The congenital cataract-causing mutations P20R and A171T are associated with important changes in the amyloidogenic feature,structure and chaperone-like activity of human αB-crystallin

Cataract is the major reason for human blindness worldwide. α-Crystallin, as a key chaperone of eye lenses, keeps the lenticular tissues in its transparent state over time. In this study, cataract-causing familial mutations, P20R and A171T, were introduced in CRYАB gene. After successful expression in Escherichia coli and subsequent purification, the recombinant proteins were subjected to extensive structural and functional analyses using various spectroscopic techniques, gel electrophoresis, and electron microscopy. The results of fluorescence and Raman assessments suggest important but discreet conformational changes in human αB-Cry upon these cataractogenic mutations. Furthermore, the mutant proteins exhibited significant secondary structural alteration as revealed by FTIR and Raman spectroscopy. An increase in conformational stability was seen in the human αB-Cry bearing these congenital cataractogenic mutations. The oligomeric size distribution and chaperone-like activity of human αB-Cry were significantly altered by these mutations. The P20R mutant protein was observed to loose most of the chaperone-like activity. Finally, these cataractogenic mutant proteins exhibited an increased propensity to form the amyloid fibrils when incubated under environmental stress. Overall, the structural and functional changes in mutated human αB-Cry proteins can shed light on the pathogenic development of congenital cataracts.     

Support for a physiological role of endogenous catecholamines in the stimulation of bovine luteal progesterone production

To determine if catecholamines were present in bovine luteal tissue, corpora lutea (CL) were obtained during the mid-luteal phase (Days 10-12) and the concentration of dopamine (DA) and norepinephrine (NE) was determined by high-performance liquid chromatography. Both DA and NE were detected in luteal tissue at mean concentrations of 41.9 +/- 5.73 and 10.2 +/- 2.51 ng/g for DA and NE, respectively. These concentrations represented a luteal content of 306.6 +/- 66.88 ng/CL for DA and 70.5 +/- 16.88 ng/CL for NE. In vitro, DA at concentrations of 1.0 mM to 0.01 mM stimulated the production of progesterone (P4, p less than 0.05). The response to DA was inhibited by propranolol (a beta-adrenergic receptor antagonist, p less than 0.05) but not by phentolamine, phenoxybenzamine (alpha-adrenergic receptor antagonists), or haloperidol (a DA receptor antagonist, p greater than 0.05). Neither L-tyrosine nor L-dopa altered P4 production (p greater than 0.05). Inhibition of DA beta-hydroxylase, the enzyme that catalyzes the conversion of DA to NE by FLA-63 blocked the DA-induced increases in luteal P4 production (p less than 0.05). These results demonstrate the existence of DA and NE in bovine luteal tissue and indicate that exogenous DA can be converted to NE in luteal tissue. The results support a physiological role for catecholamines in the stimulation of bovine luteal function.     

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: I. Fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases

The phase transitions in fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases have been studied by lowangle time-resolved x-ray diffraction under conditions similar to those employed in calorimetry (scan rates 0.05-0.5°C/min and uniform temperature throughout the samples). This approach provides more adequate characterization of the equilibrium transition pathways and allows for close correlations between structural and thermodynamic data. No coexistence of the rippled gel (P_β') and liquid-crystalline (L_α) phases was found in the main transition of DPPC; rather, a loss of correlation in the lamellar structure, observed as broadening of the lamellar reflections, takes place in a narrow temperature range of ~100 mK at the transition midpoint. Formation of a long-living metastable phase, denoted by P_β'(mst), differing from the initial P_β' was observed in cooling direction by both x-ray diffraction and calorimetry. No direct conversion of P_β'(mst) into P_β' occurs for over 24 h but only by way of the phase sequence P_β'(mst) → L_β' → P_β'. According to differential scanning calorimetry (DSC), the enthalpy of the P_β'(mst)-L_α transition is by ~5% lower than that of the P_β'-L_α transition. The effects of ethanol (Rowe, E. S. 1983. Biochemistry. 22:3299-3305; Simon, S. A., and T. J. McIntosh. 1984. Biochim. Biophys. Acta 773:169-172) on the mechanism and reversibility of the DPPC main transition were clearly visualized. At ethanol concentrations inducing formation of interdigitated gel phase, the main transition proceeds through a coexistence of the initial and final phases over a finite temperature range. During the subtransition in DPPC recorded at scan rate 0.3°C/min, a smooth monotonic increase of the lamellar spacing from its subgel (L_c) to its gel (L_β') phase value takes place. The width of the lamellar reflections remains unchanged during this transformation. This provides grounds to propose a “sequential” relaxation mechanism for the subgel-gel transition which is not accompanied by growth of domains of the final phase within the initial one.     

Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationship among different serovars of the Mycobacterium avium-M. intracellulare complex as well as to define signature nucleotides characteristic for different serovars. In general, the groups defined by rRNA sequencing reflect the classification obtained with sensitin tests and pathogenicity examinations in chickens. Unique 16S rRNA sequence patterns could be defined for (1) M. avium, (2) M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11, (3) M. intracellulare serovars 12, 13, 14, 15, 17, 19 and 20, (4) M. intracellulare serovar 7 and (5) M. intracellulare serovar 18. Phylogenetically, groups 1 and 2 on one hand and groups 3, 4 and 5 on the other hand each share a common ancestor. M. paratuberculosis was indistinguishable from M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11 by this kind of analysis.     

Size-dependent drift responses of mayflies to experimental hydrologic variation: active predator avoidance or passive hydrodynamic displacement?

Summary Larger nymphs within aquatic insect taxa have been frequently observed to be transported down-stream in the stream drift only at night. Others have hypothesized this pattern results primarily from large nymphs' behavioural avoidance of entering drift during daylight, when size-selective, visually-feeding fish predators are most active. This hypothesis assumes that animals can actively control their entry into the drift, which may not be the case under all flow conditions. We experimentally induced streamflow increases and decreases in adjacent riffles in a hydrologically-stable stream during the daytime to examine whether changes in diel patterns of drift abundance and size-distribution of mayflies were consistent with the hypothesis of active avoidance of diurnal drift. We assessed the likelihood of active vs. passive mechanisms of diurnal drift entry and transport for four taxa that differ with respect to body size, morpho-behavioural attributes, microhabitat use, and general propensity to drift. In each of three seasons, diurnal and nocturnal drift samples were collected in three riffles over two diel cycles. Background drift patterns were established on the first day (no flow manipulation). Six h before sunset on the second day, flow was experimentally increased in one riffle, decreased in the second, and not altered in the third (control). Between-day differences in diurnal and nocturnal drift rate and size composition were then compared among the treatment and reference riffles. Responses of two taxa were consistent with active control over drift entry, transport, or both. For Baetis spp., drift-prone mayflies typically preyed upon by fish, diurnal drift rates immediately increased following both flow reduction and flow elevation in all seasons, but only small individuals comprised the drift. Drift by large individuals was delayed until nighttime. Epeorus longimanus also exhibited significant increases in drift rates following flow reduction and elevation, but responses of this large-bodied species were restricted to nighttime. Drift responses for these two taxa were largely independent of direction of hydrologic change, thus indicating a strong behavioural control over drift. By contrast, numbers and sizes of drifting Paraleptophlebia heteronea and Ephemerella infrequens depended strongly on direction of flow change. Drift rates for both species generally declined after flow reduction and increased after flow elevation. Moreover, after flow elevation, larger individuals often drifted diurnally, a finding consistent with expectations under a passive hydrodynamic model. These experiments indicate that size-dependent mayfly drift reflects not only presumed risk from visual fish predators, but also functional attributes of species such as morphology, behaviour, and microhabitat affiliation, which influence aspects of drift entry and transport under variable hydrologic conditions.     

Alteration of Striatal Glutamate Release After Glutamine Synthetase Inhibition

The effect of the glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), on glutamate levels in, and glutamate release from, rat striatal tissue was examined. Tissue levels of glutamate were unchanged 24 h after an intraventricular injection of MSO, but tissue glutamine levels were decreased 50%. Calcium-dependent, potassium-stimulated glutamate release was diminished in tissue prisms from animals pretreated with MSO compared to controls. The decreased release of glutamate correlated over time with the inhibition of GS following an intraventricular injection of MSO. The maximum diminution of calcium-dependent, potassium-stimulated glutamate release (50%) and the maximum inhibition of GS activity (51%) were observed 24 h after MSO. The addition of 0.5 mM glutamine to the perfusion medium completely reversed the effects of MSO pretreatment on calcium-dependent, potassium-stimulated glutamate release. Since GS is localized in glial cells and the measured glutamate release is presumed to occur from neurons, the data support the contention that astroglial glutamine synthesis is an important contributor to normal neuronal neurotransmitter release.