Phosphorylation of iodopsin, chicken red-sensitive cone visual pigment

The amino acid sequence has been determined for the carboxyl-terminal 41 amino acids of chicken red-sensitive cone pigment, iodopsin. This sequence is distinct from but structurally homologous to that of other visual pigments. It contains a region rich in the hydroxy amino acids serine and threonine. In the related rod cell visual pigment, rhodopsin, such serines and threonines have previously been identified as sites for phosphorylation by rhodopsin kinase. Phosphorylation of photolyzed rhodopsin serves to terminate its ability to function in visual transduction as an activator of G-protein. We have purified and reconstituted both chicken rhodopsin and chicken iodopsin and shown them to be phosphorylated by bovine rhodopsin kinase. Chicken iodopsin has a Km and Vmax similar to but distinguishably different from that for bovine rhodopsin. These results, in conjunction with other data, suggest that visual pigments in cone cells, upon absorption of light, undergo functional processes similar to those of the visual pigments in rod cells.     

The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome’s efferent arm) make fragments after less heat than controls. If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the “baggage” (nucleus, granuloplasm)—i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.     

The binding of adenosine to polyuridylic acid in which uracil residues are chemically altered.

The binding of adenosine-¹⁴C to polyuridylic acid (poly(U)) and several modified poly(U)s has been studied by equilibrium dialysis. The poly(U) was modified by addition of appropriate reagents across the 5,6-double bond of the uracil ring to form the photohydrate, photodimer, dihydrouracil, the HOBr addition product and the HSO₃^? addition product. Modification of the uracil rings decreases the amount of adenosine which can be bound to the poly(U); the decrease in binding is a function of the fraction of uracil rings which have been changed. Using the expression S = S₀(1 ? αr)² to relate the fraction of uracil rings modified (r) to the number of binding “sites” remaining (S), it is found that α is about 1 for all the modifications except photodimer where it is about 2. These observations are taken to mean that the loss of binding capacity of the poly(U) resulting from modifications of the uracil ring is caused by loss of planarity of the uracil rings caused by the modifications, and consequent loss of double helix structure, but that for all modifications except photodimer there is no disruption of the poly(U) double helix on either side of the leison. There does appear to be local melting on either side of the photodimer lesion. The sigmoidal binding isotherms (A_b versus C_a) of modified and unmodified poly(U) can be approximated closely by the following equation: ((1)) (1) where A_b = bound A, C_a = free A, n = minimum number of adjacent A′s in complex, S = concentration of sites on poly(U), and K₁ = (K_m)^1/m for all m ≥ n. The stacking energy of adenosine (w) can be calculated accurately using the following equation, where dθ/d ln C_a is obtained from Eq. (1). ((2)) (2) For unmodified poly(U), w is ?2.0 kcal/mole and ΔG° (?;RT ln K₁) is ?3.2 kcal/mole. The difference (?1.2 kcal/mole) is attributed to hydrogen bonding. Heavily photohydrated poly(U) does not bind guanosine or guanosine-5′-phosphate.     

A comparison of immunomagnetic and surface adhesion immunofluorescent techniques for the rapid detection of Listeria monocytogenes and Listeria innocua in meat

an immunomagnetic immunofluorescent method was investigated for the rapid detection of Listeria monocytogenes and Listeria innouca . This technique involved enrichment of the suspect sample at 30°C overnight. Listeria monocytogenes cells were isolated from the enriched sample using immunomagnetic separation and Listeria were subsequently visualized using an immunofluorescent microscopy technique. This technique was used in the detection of Listeria cells from pure culture, inoculated beef mince samples and naturally contaminated retail beef mince samples. A detection level of approximately 1×10³ cfu ml⁻¹ was achieved. When compared with traditional detection methods no false negatives or positives were recorded for L. monocytogenes or L. innocua . The immunomagnetic immunofluorescent technique had a detection level similar to a previously described surface adhesion immunofluorescent technique. Isolation of the Listeria cells by surface adhesion involved dipping a membrane attached to a microscope slide into the enriched sample for 10 min. This was quicker and simpler to perform than the immunomagnetic separation technique which took 2 h to carry out.     

Correlations between blood glucose levels and bromodeoxyuridine labelling indices of pancreatic islet cells following streptozotocin administration to pregnant Syrian golden hamsters

Eighteen timed-pregnant Syrian golden hamsters were injected subcutaneously with streptozotocin (STZ, 60 mg/kg bw) early on gestational day 10. The response varied widely, and based on changes in blood glucose levels during gestational days 11 to 15, the hamsters were categorized into four groups: 1) no change; 2) mild diabetes (200-250 mg/dl), which reverted; 3) moderate diabetes (greater than 300 mg/dl), which reverted; and 4) moderate to severe diabetes (300-500 mg/dl), which was sustained. Two hours before sacrifice, a 25 mg tablet of bromodeoxyuridine (BrdU) was implanted subcutaneously into each experimental hamster and into 17 control pregnant hamsters that had not received STZ. BrdU-labelling was demonstrated immunochemically in the pancreatic islet cells. In control hamsters, the mean labelling index (LI) of the islet cells was 0.07% and did not exceed 0.2% in any hamster. Following injection of STZ, islet cell LI's remained low (0.13%) if the blood glucose levels were not altered by the diabetogenic drug. However, LI's were increased in islet cells of hamsters which showed a mild to moderate diabetes which rapidly reverted; the highest LI's (5% +/- 2.1) occurred in four hamsters that were killed 2 days after receiving STZ. The LI's were moderately increased (1.4% +/- 0.42) in two hamsters with moderate diabetes killed 2 days after STZ, but LI's were low (0.12% +/- 0.04) in six hamsters with moderate to severe diabetes killed 3, 4, and 5 days after STZ. Reversion of hyperglycemia to normoglycemia correlated closely with increased DNA synthesis in the islet cells of the pregnant hamsters. These observations strongly suggest that following mild cytotoxic injury induced by STZ, the B cells regenerated and insulin production was restored sufficiently to maintain normoglycemia.     

Preparation of Peptides Containing Any Desired Amino Acid: Methionyl Peptides of Bovine Rhodopsin

A general method is described which allows the identification and preparation of peptides containing any amino acid of interest. The method has been applied to isolation of the methionyl peptides from a peptic digest of oxidized bovine rhodopsin. The peptide digestion mixture is first partially separated by ion exchange column chromatography. Location of peptides containing the desired amino acid is performed by amino acid analysis of acid hydrolyzed column fractions by high voltage paper electrophoresis. Peptides are further purified and prepared by peptide mapping, elution, and amino acid analysis using inexpensive high capacity techniques. Peptide sequencing is performed by a manual dansyl-Edman method well adapted for rapidly processing large numbers of samples. The methods are particularly well suited for detection and preparation of peptides containing amino acids for which there is no specific detection method.     

When Wet Gets Wetter: Decoupling of Moisture, Redox Biogeochemistry, and Greenhouse Gas Fluxes in a Humid Tropical Forest Soil

Upland humid tropical forest soils are often characterized by fluctuating redox dynamics that vary temporally and spatially across the landscape. An increase in the frequency and intensity of rainfall events with climate change is likely to affect soil redox reactions that control the production and emissions of greenhouse gases. We used a 24-day rainfall manipulation experiment to evaluate temporal and spatial trends of surface soil (0–20 cm) redox-active chemical species and greenhouse gas fluxes in the Luquillo Experimental Forest, Puerto Rico. Treatments consisted of a high rainfall simulation (60 mm day^?1), a fluctuating rainfall regime, and a control. Water addition generated high temporal and spatial variation in soil moisture (0.3–0.6 m³ m^?3), but had no significant effect on soil oxygen (O₂) concentrations. Extractable nitrate (NO₃ ^?) concentrations decreased with daily water additions and reduced iron (Fe(II)) concentrations increased towards the end of the experiment. Overall, redox indicators displayed a weak, non-deterministic, nonlinear relationship with soil moisture. High concentrations of Fe(II) and manganese (Mn) were present even where moisture was relatively low, and net Mn reduction occurred in all plots including controls. Mean CO₂ fluxes were best explained by soil C concentrations and a composite redox indicator, and not water addition. Several plots were CH₄ sources irrespective of water addition, whereas other plots oscillated between weak CH₄ sources and sinks. Fluxes of N₂O were highest in control plots and were consistently low in water-addition plots. Together, these data suggest (1) a relative decoupling between soil moisture and redox processes at our spatial and temporal scales of measurement, (2) the co-occurrence of aerobic and anaerobic biogeochemical processes in well-drained surface soils, and (3) an absence of threshold effects from sustained precipitation on redox reactions over the scale of weeks. Our data suggest a need to re-evaluate representations of moisture in biogeochemical models.     

The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death

Components of the vesicle trafficking machinery are central to the immune response in plants. The role of vesicle trafficking during pre-invasive penetration resistance has been well documented. However, emerging evidence also implicates vesicle trafficking in early immune signaling. Here we report that Exo70B1, a subunit of the exocyst complex which mediates early tethering during exocytosis is involved in resistance. We show that exo70B1 mutants display pathogen-specific immuno-compromised phenotypes. We also show that exo70B1 mutants display lesion-mimic cell death, which in combination with the reduced responsiveness to pathogen-associated molecular patterns (PAMPs) results in complex immunity-related phenotypes.     

Mild Transient Hypercapnia as a Novel Fear Conditioning Stimulus Allowing Re-Exposure during Sleep

Introduction

Studies suggest that sleep plays a role in traumatic memories and that treatment of sleep disorders may help alleviate symptoms of posttraumatic stress disorder. Fear-conditioning paradigms in rodents are used to investigate causal mechanisms of fear acquisition and the relationship between sleep and posttraumatic behaviors. We developed a novel conditioning stimulus (CS) that evoked fear and was subsequently used to study re-exposure to the CS during sleep.

Methods

Experiment 1 assessed physiological responses to a conditioned stimulus (mild transient hypercapnia, mtHC; 3.0% CO₂; n = 17)+footshock for the purpose of establishing a novel CS in male FVB/J mice. Responses to the novel CS were compared to tone+footshock (n = 18) and control groups of tone alone (n = 17) and mild transient hypercapnia alone (n = 10). A second proof of principle experiment re-exposed animals during sleep to mild transient hypercapnia or air (control) to study sleep processes related to the CS.

Results

Footshock elicited a response of acute tachycardia (30–40 bpm) and increased plasma epinephrine. When tone predicted footshock it elicited mild hypertension (1–2 mmHg) and a three-fold increase in plasma epinephrine. When mtHC predicted footshock it also induced mild hypertension, but additionally elicited a conditioned bradycardia and a smaller increase in plasma epinephrine. The overall mean 24 hour sleep–wake profile was unaffected immediately after fear conditioning.

Discussion

Our study demonstrates the efficacy of mtHC as a conditioning stimulus that is perceptible but innocuous (relative to tone) and applicable during sleep. This novel model will allow future studies to explore sleep-dependent mechanisms underlying maladaptive fear responses, as well as elucidate the moderators of the relationship between fear responses and sleep.     

Dynamics of arrestin-rhodopsin interactions: loop movement is involved in arrestin activation and receptor binding

In this study we investigate conformational changes in Loop V-VI of visual arrestin during binding to light-activated, phosphorylated rhodopsin (Rho*-P) using a combination of site-specific cysteine mutagenesis and intramolecular fluorescence quenching. Introduction of cysteines at positions in the N-domain at residues predicted to be in close proximity to Ile-72 in Loop V-VI of arrestin (i.e. Glu-148 and Lys-298) appear to form an intramolecular disulfide bond with I72C, significantly diminishing the binding of arrestin to Rho*-P. Using a fluorescence approach, we show that the steady-state emission from a monobromobimane fluorophore in Loop V-VI is quenched by tryptophan residues placed at 148 or 298. This quenching is relieved upon binding of arrestin to Rho*-P. These results suggest that arrestin Loop V-VI moves during binding to Rho*-P and that conformational flexibility of this loop is essential for arrestin to adopt a high affinity binding state.