Fluorescence Resonance Energy Transfer between Lipid Probes Detects Nanoscopic Heterogeneity in the Plasma Membrane of Live Cells
Fluorescence resonance energy transfer (FRET) between matched carbocyanine lipid analogs in the plasma membrane outer leaflet of RBL mast cells was used to investigate lateral distributions of lipids and to develop a general method for quantitative measurements of lipid heterogeneity in live cell membranes. FRET measured as fluorescence quenching of long-chain donor probes such as DiO-C^sub 18^ is greater with long-chain, saturated acceptor probes such as DiI-C^sub 16^ than with unsaturated or shorter-chain acceptors with the same chromophoric headgroup compared at identical concentrations. FRET measurements between these lipid probes in model membranes support the conclusion that differential donor quenching is not caused by nonideal mixing or spectroscopic differences. Sucrose gradient analysis of plasma membrane-labeled, Triton X-100-lysed cells shows that proximity measured by FRET correlates with the extent of lipid probe partitioning into detergent-resistant membranes. FRET between DiO-C^sub 16^ and DiI-C^sub 16^ is sensitive to cholesterol depletion and disruption of liquid order (Lo) by short-chain ceramides, and it is enhanced by cross linking of Lo-associated proteins. Consistent results are obtained when homo-FRET is measured by decreased fluorescence anisotropy of DiI-C^sub 16^. These results support the existence of nanometer-scale Lo/liquid disorder heterogeneity of lipids in the outer leaflet of the plasma membrane in live cells.
The existence of lateral inhomogeneities or domains in the lipid portion of the plasma membrane is an issue of substantial interest and controversy . Studies on detergent-resistant membranes led to the hypothesis that lateral segregation of liquid ordered (Lo) and liquid disordered (Ld) lipids in the plasma membrane plays an important role in signal transduction, protein sorting, and membrane transport . These cholesterol-dependent ordered membrane domains that selectively contain lipids and proteins are commonly called lipid rafts. However, the difficulty in observing lipid membrane domains at optical resolution has made it challenging to relate biochemical evidence for lipid heterogeneity, such as that from detergent-dependent fractionation, to properties in live cells. Detecting segregated membrane domains in live cells usually requires large-scale cross-linking of lipid raft components and/or low temperature (7-9). Some advanced methods with high spatial (nanometer) and temporal (millisecond) resolution, such as single particle tracking and video microscopy, nanosecond depolarization homo-FRET (13), and high-resolution electron microscopy , have provided evidence for membrane domains in intact plasma membranes. However, direct evidence for the existence of lipid rafts in live cells is largely based on measurements of clustering or diffusion of putative raft proteins rather than on measurements of the lipids themselves. A recent exception is the detection of the ordered and disordered lipid phases in live cells with ESR measurements of spin-labeled lipid probes (16).The ordered state of lipid rafts is based on preferential interactions between cholesterol and phospholipids with long saturated acyl chains such as sphingolipids. Correspondingly, lipid probes with different acyl/alkyl chains are expected to partition differently between ordered domains and disordered regions of the bilayer. In our study, we investigated the lateral distributions of fluorescent lipid probes in the outer leaflet of live cell membranes using FRET between carbocyanine derivatives with differing alkyl chains. Carbocyanines label the outer leaflet of cell membrane with negligible transbilayer flip-flop (17), making them ideal outer leaflet probes. Furthermore, their high photostability, large extinction coefficients, and partition coefficients that strongly favor lipid over aqueous environments (K^sub p^ ~ 10^sup 3-4^ for DiO-C^sub 6^) (18) make them highly suitable for fluorescence measurements in live cell membranes. FRET is particularly useful for monitoring inhomogeneities in the lateral organization of lipid bilayers on a spatial scale intermediate between the ~300-nm scale of light microscopy and the nearest neighbor (0.1 nm) scale of Stokes quenching of fluorescent or brominated lipids by spin labels (19,20). Previous measurements of FRET between lipidanchored fluorescent proteins at the inner leaflet of plasma membranes provided evidence for inhomogeneity on the scale of tens of nanometers (21), illustrating the value of this noninvasive technique for studying membrane domains with dimensions below optical resolution.
Our measurements of FRET between carbocyanine lipid probes in the plasma membrane of live cells provide strong evidence for lateral lipid inhomogeneities that are sensitive to perturbations that enhance or reduce Lo/Ld segregation. These results correlate well with lipid partitioning as measured by detergent resistance. The method we describe provides a novel approach to studying membrane lipid heterogeneity in live cells that is relevant to cell functions.
A Three-Stage Kinetic Model of Amyloid Fibrillation
Amyloid fibrillation has been intensively studied because of its association with various neurological disorders. While extensive time-dependent fibrillation experimental data are available and appear similar, few mechanistic models have been developed to unify those results. The aim of this work was to interpret these experimental results via a rigorous mathematical model that incorporates the physical chemistry of nucleation and fibril growth dynamics. A three-stage mechanism consisting of protein misfolding, nucleation, and fibril elongation is proposed and supported by the features of homogeneous fibrillation responses. Estimated by nonlinear least-squares algorithms, the rate constants for nucleation were ~10,000,000 times smaller than those for fibril growth. These results, coupled with the positive feedback characteristics of the elongation process, account for the typical sigmoidal behavior during fibrillation. In addition, experiments with different proteins, various initial concentrations, seeding versus nonseeding, and several agitation rates were analyzed with respect to fibrillation using our new model. The wide applicability of the model confirms that fibrillation kinetics may be fairly similar among amyloid proteins and for different environmental factors. Recommendations on further experiments and on the possible use of molecular simulations to determine the desired properties of potential fibrillation inhibitors are offered.
Amyloid fibrillation is the process of native soluble proteins misfolding into insoluble fibrils comprising cross-?-sheets. More than 20 amyloidogenic diseases such as Alzheimer’s disease, Parkinson’s disease, and prion-associated encephalopathies have been found to share fibril formation as the common symptom (1). The presence of amyloid plaques correlates with disease, but whether fibrils themselves, misfolded oligomers, or other factors are the causal agents of diseases remains unclear . Although the proteins associated with each disease do not share sequence homology, they exhibit similar insoluble filaments and fibrillation responses . This suggests that the underlying fibril formation mechanisms may be common (7).
The typical fibril formation process starts with a lag phase in which the amount of amyloid proteins turned into of fibrils is not significant enough to be detected. Afterwards, a drastic elongation phase follows and fibril concentration increases rapidly (8). Eventually, the process reaches equilibrium when most soluble proteins are converted into fibrils. The length of lag times and fibril growth rates depend upon factors like the initial concentration and pH, both of which affect the degree of supersaturation in solution. The presence of seeded molecules and foreign surfaces can influence the kinetics of fibrillation, because of the ability to catalyze the reactions at these interfaces (9). Other factors include the ionic strength of the solution and the intensity of agitation (10). Although experimental data covering these many different conditions have been reported in the literature, there is a noticeable lack of quantitative mechanistic models to provide insight into the process and directions for further research.
Because of the commonly observed sigmoidal-shaped fibrillation response reported in the literature, fibrillation processes have been modeled as a number of reactions in series covering the assembly of oligomers, the formation of nuciei as well as the growth and the breakage of fibrils. Moreover, the two-stage mechanism of yeast prion fibrillation, in which fibrils act as enzymes to trigger nucleated conformational conversion by Michaelis-Menten kinetics, provides another valuable perspective (14). Empirical or semi-empirical exponential functions are popular choices to fit the data since they are computationally simple and match the observed data well . While suggestive, some of these models only depicted the sigmoidal trend without rigorous quantitative arguments; others have not provided details on how the nuclei form or explained the shortened lag-time resulting from seeding and an increase in the initial protein concentration.
The lag-time before fibril growth has been noted in numerous publications and resembles an incubation period (10,11). Explaining its existence is one of the key scientific challenges. The problem was approached by Shoghi-Jadid et al. (16) with introduction of the Heaviside function to force the separation of nucleation and fibrillation processes, while Uversky et al. (17) used an empirical exponential model with adjustable parameters. We suggest that nucleation theory and growth models could be valuable in describing the fibrillation process. Furthermore, the drastic rate increase in the fibrillar growth phase after the lag phase indicates that cooperativity or positive feedback mechanisms are involved.
Another critical but missing piece of information is the relationship between the observable response and the degree of fibrillation. Even though histological dyes like thioflavin T (ThT) and Congo Red have been the commonly used as indicators of the presence of amyloid fibrils, the relationship between fluorescence intensity and amount of amyloid fibril remain unclear. There are also physical property methods for measuring fibril formation like turbidity, absorbance, and sedimentation . Here, we assumed linearity between ThT fluorescence and fibril concentrations based on Beer-Lambert law as a measure of fibril content, and use ultraviol et-visible (UV-vis) absorbance at 280 nm as a quantitative measure of dissolved total protein.
Insulin was chosen as the model protein for the measurements in this study because it 1), is a well-studied fibril-forming protein and has recently been studied in our laboratory, is known to develop structurally similar cross-?-sheet plaques to those formed by other amyloids and is deposited in arterial walls of type II diabetes patients (23); and 4), is available in large quantities at reasonable price. Native insulin is well folded and in stable hexamer state associated with Zn^sup 2+^ molecule under physiological conditions. Yet it can be readily unfolded to form fibrils in solution by both increasing the temperature to 65°C and by reducing the pH to 1.6. Jiménez et al. (28) proposed that the ?-helical structure (58%) of native insulin becomes unfolded to expose the ?-sheet region (6%), which is the major component of the amyloid cross-? ribbon.
In the next section, we describe the proposed kinetic model for insulin fibrillation including the parameter estimation procedure. Since experimental protocols and responses of fibrillation are similar among amyloid proteins, the modeling approach presented here is also applicable to the fibrillation of other proteins. Afterwards, our model is compared with an empirical fitting function. A general description of the Experimental Materials and Methods follows. Then, in Results and Discussion, the new model is fitted to our insulin fibrillation data, to fibrillation of A?-40 and prionlike NM fragment of Sup-35 , and to data conducted under various conditions .
Three standard analytical steps were chosen to model insulin fibrillation; formulation of the appropriate kinetic reactions based on the polymerization and nucleation theories, conversions of the reaction set into a system of differential equations, and parameter estimation by nonlinear least-square algorithms to optimize the fit between simulation results and the experimental measurements.
Initially four species of insulin were considered during fibrillation: original hexamer, monomer, cluster, and fibril . While the original hexamer is composed of six monomers stabilized by Zn^sup 2+^ an insulin monomer refers to two chains of polypeptides connected with disulfide bonds . For systems other than insulin, different morphologies may be involved such as those for ?-microglobulin (26). By incorporating the four insulin species into the reaction scheme, the proposed kinetic mechanism for this study consists of three distinct stages: decomposition of hexamers, nucleation process, and fibrillation stage as summarized in Fig. 1 and Table 1. All the reactions listed are elementary reactions so the fluxes can be easily expressed as the products of reactant concentrations and the rate constant. Regarding notations, A^sub hex^ and A^sub i^ denote the concentration of original insulin hexamers and oligomers containing i monomers, respectively. All fibrils are abbreviated as F, regardless of their length. Even though physical reactions contributing to larger-size cluster formation and the entanglement between strands of fibrils have been reported , the actual active chemical reaction sites are assumed to be restricted to the fibril ends . Therefore, fibrils of different sizes can be considered as the same species.
Sclerosing Angiomatoid Nodular Transformation of the Spleen
Sclerosing angiomatoid nodular transformation (SANT) is a recently recognized nonneoplastic vascular lesion of the spleen with fewer than 30 cases described. Microscopically, SANT consists of multiple well-circumscribed vascular/ angiomatoid nodules showing plump endothelial cell and extravasated erythrocytes. The nodules are surrounded by a variable lymphoplasmacytic infiltrate, spindle cells, and collagenous stroma. The vascular nodules display a complex mixture of endothelial phenotypes resembling splenic sinusoids (CD34^sup -^/CD31^sup +^/CD8^sup +^), capillaries (CD34^sup +^/ CD31^sup +^/CD8^sup -^), and small veins (CD34^sup -^/CD31^sup +^/CD8^sup -^). Focal expression of CD68 can also be seen. The differential diagnosis of SANT includes splenic hamartoma, inflammatory myofibroblastic tumor, littoral cell angioma, and hemangioendothelioma. It has been postulated that SANT represents a peculiar hamartomatous transformation of splenic red pulp in response to an exaggerated nonneo-plastic stromal proliferation. SANT has a benign clinical course with splenectomy being curative.
Hemangiomas are the most common primary tumors of the spleen.1 Other rare vascular splenic lesions include lymphangioma,2 littoral cell angioma (LCA),3 he-mangioendothelioma, 4 and splenic hamartoma.5 In 2004 Martel et al6 described a distinctive nonneoplastic vascular lesion of the spleen, which they designated sclerosing an-giomatoid nodular transformation (SANT). Isolated examples of SANT had been previously described as splenic hamar-toma7 and hemangioendothelioma.8 In this review, we describe the clinical and pathologic features of SANT and discuss its major differential diagnoses.
Sclerosing angiomatoid nodular transformation is rare. In their original description, Martel et al6 reported 25 cases and another example was later added to the literature.9 Recently we have seen 2 cases affecting middle-aged women. The mean age at presentation is 53.7 years with a range from 22 to 74 years. Sclerosing angiomatoid nodular transformation shows a marked female predominance with a female-male ratio of 2:1.6,9 In the series by Martel et al,6 most patients were asymptomatic and the splenic mass was an incidental finding during laparotomy or during imaging studies for unrelated conditions. Some patients- approximately 16%-complained of abdominal pain or discomfort. A minority of patients presented with splenomegaly rarely associated with leukocytosis, poly-clonal gammopathy, and increased erythrocyte sedimentation rate.6 Reported concurrent diseases include hypertension, von Willebrand disease, chronic lymphocytic leukemia, and carcinomas of lung, gastrointestinal tract, and kidney with no metastasis to the spleen.6 The patient reported by Li et al9 had multiple medical problems including hypertension, diabetes mellitus, hypothyroidism, and prostate hyperplasia. Abdominal ultrasound, computed tomography scans, and magnetic resonance imaging studies usually reveal a hypodense, multinodular splenicmass. Computed tomography or magnetic resonance imaging with contrast cannot distinguish SANT from the surrounding spleen parenchyma.9 Splenectomy is usually performed on the discovery of a splenic mass and appears to be curative in all reported cases.
Although the histologic and immunohistochemical features of SANT have been characterized only recently by Martel et al,6 isolated cases were described as early as 19787 under the terms splenic hamartoma,5,7 cord-capillary hemangioma,10 multinodular hemangioma,11 and as a variant of splenic hemangioendothelioma.8 At low-power magnification, SANT is composed of multiple well-circumscribed individual and confluent vascular/angiomatoid nodules
with a variable fibrosclerotic stroma . The nodules are round to oval with variable sizes. Some are surrounded by dense concentric collagen fibers , whereas others show a fibrin rim resulting in a granuloma-like or necrotizing vasculitic appearance . Each nodule demonstrates a mixture of slit-like vascular spaces with a sievelike appearance lined by plump endothelial cells and pericytes admixed with ex-travasated red blood cells . The vessels incorporated in the nodules are highlighted by a reticulin stain . The endothelial cells show minimal cellular atypia, and only rare mitotic activity is seen within the nodules. The internodular stroma is fibromyxoid or sclerotic and contains variable numbers of myofibroblasts, plasma cells, lymphocytes, macrophages, and hemosiderin laden macrophages . The intercellular stro-ma may show large areas of hyalinization . The adjacent splenic tissue is usually compressed by the nodules but is otherwise within normal limits.
Serum sickness-like reaction to cefuroxime: a case report and review of the literature
We report a case of a 34-year-old woman who received cefuroxime, a second-generation cephalosporin, as treatment for mastitis. She subsequently developed a serum sickness-like reaction (SSLR) with a generalized pruritic rash, joint pains, and fever. She improved upon treatment with systemic steroids. SSLR is well-described to cefaclor, a second-generation cephalosporin. However, there is a paucity of reports of SSLR to other cephalosporins such as this case.
A 34-year-old Caucasian woman presented to the dermatology clinic with a 2-week history of mastitis. The patient was breast-feeding following delivery of her sixth child 7 weeks earlier. She had no other significant past medical history. She was treated with 500 mg of oral cefuroxime twice daily and had completed the 10-day prescribed course. Six days into the course of treatment, she developed a rash on the scalp, which generalized over a 5-day time span to involve extensive areas of the trunk, arms, legs, and face. The rash was very pruritic, and individual lesions, which lasted less than 24 hours, were migratory. The patient also complained of intermittent fevers to 101F and joint pains of the elbows, knees, and smaller joints of the hands. She specifically denied wheezing or shortness of breath. Her other current medications included prenatal vitamins. At the time of her delivery, 7 weeks previous, she had received pain medications, but no antibiotics. Acetaminophen and diphenhydramine, which she had tolerated well in the past, were taken orally as needed for the rash. Her reported allergies included tetracycline, penicillin, and erythromycin, all of which had caused skin rashes. As the rash continued to worsen in extent and severity, even after the antibiotic was completed, she was treated with oral methylprednisolone for 3 days, then one dose of oral prednisone 30 mg without improvement. She was then referred to the dermatology clinic.
The patient was a thin woman who scratched frequently, but otherwise was in no apparent distress. Skin examination revealed multiple confluent and extensive blanching urticarial plaques with dermatographism on the arms, legs, trunk, and face. No mucosal erosions were present, and there was no edema of the face or extremities. No cervical, occipital, axillary, or inguinal lymphadenopathy was noted. Pulmonary and cardiac examination was unremarkable. She had no swelling or erythema of the joints. The elbows, knees, and joints of the hands exhibited full range of motion. Laboratory studies, including a complete blood count, comprehensive metabolic panel, urinalysis, erythrocyte sedimentation rate, and C3, C4, and CH50 were all within the normal limits.
She was diagnosed with a serum sickness-like reaction (SSLR) to cefuroxime and admitted for 100 mg of intravenous methylprednisolone daily and oral antihistamines. Her symptoms and rash improved within 24 hours, and she was discharged on an extended tapering dose of prednisone starting at 40 mg daily, in addition to oral hydroxyzine, oral doxepin, and topical emollients. She experienced slow but steady resolution of her symptoms over the following 4 weeks.
SSLR is a specific type of drug reaction so named because of its clinical similarity to serum sickness. True serum sickness is a type III hypersensitivity reaction in which clinical signs and symptoms result from deposition of immune complexes in the skin, joints, and other organ systems. In contrast, SSLR is not associated with demonstrable circulating immune complexes. The reaction is acute, self-limited, and has been described in association with a variety of different medications. Although many drugs have been reported to cause SSLR, antibiotics are the major group of offending agents, particularly beta-lactam and sulfonamide antibiotics.
In most cases, signs and symptoms appear about a week after initiation of therapy. (3) The most frequent finding is cutaneous involvement, typically in the form of erythema and urticarial lesions that are often migratory. In the series reported by Hebert et al, many of the urticarial wheals had dusky to purple centers, which were morphologically suggestive of erythema multiforme (EM).
The other primary clinical feature is joint involvement. Pain and swelling of the joints is a typical finding, and is usually polyarticular. Affected joints commonly include wrists, ankles, hips, and knees, which can become so severe that patients are unable to walk. (2)
Although fever may occur, other systemic findings are less common. In contrast to true serum sickness, renal and hepatic involvement is rare.
Despite the impressive cutaneous findings and joint symptoms, a hallmark of SSLR is the benign outcome, although some patients do require hospitalization due to their severe symptoms. Treatment is typically symptomatic, usually with antihistamines and analgesics. The use of systemic corticosteroid treatment has been described in retrospective medical record reviews (4) and in a number of case reports, although there is no accepted standardized therapy. Reports of SSLR describe benign outcomes with no sequelae.
Combined tide and wave influence on sedimentation of Lower Gondwana coal measures of central India: Barakar Formation , Satpura basin
Abstract:The coal-bearing Barakar succession of the Satpura basin is typical of the Gondwanan coal basins of peninsular India, in that it has previously been interpreted as continental in origin. The succession comprises three main facies associations, which are documented within this paper. Medium- to fine-grained muddy sandstone deposits of 5-75 m thickness are reinterpreted as tidally influenced delta deposits. Mudstone deposits of 3-20 m thickness with subordinate sandstones, coals and carbonaceous shales are reinterpreted as delta top deposits, and medium to coarse sandstones of 3-38 m thickness are interpreted as braided delta-top channels. The evidence for tidal influence arises from documentation of bidirectional cross-strata, tidal bundles, tidal rhythmites and periodic variation in foreset thickness. The recognition of tidal deposits indicates marine depositional conditions and significantly changes existing palaeogcographical models. This in turn has important implications for our understanding of the depositional setting and distribution of the Permian coals that occur across much of the southern supercontinent. Furthermore, to the best of the authors’ knowledge, coal-bearing tidal-delta deposits have not previously been described from continental interior basins.
The Permian strata of the coal-bearing Barakar Formation of the Satpura Gondwana basin, India were deposited in an intracontinental basin and have previously been ascribed a non-marine origin based on the absence of marine fossils, and the similarities of some of the sediment bodies to fluvial deposits . Accordingly, it has been suggested by most previous studies that the Satpura basin was geographically restricted from oceanic waters and that tidal reworking had no effect on the deposition of the Barakar strata . Although marine fossils have not been recognized, possible marine influence during Barakar sedimentation has been proposed by several workers based on the presence of wave-generated structures in the sandstones, trace fossils and high boron contents of the coal . However, wave-influenced deposits may also form in exclusively continental domains , and their formation in a marine realm can only be ascertained if they are associated with tide-influenced deposits . Recognition of tidal signatures in strata affected also by fluvial processes is not as straightforward as in open marine strata . This paper describes combined tide-and wave-influenced facies from the Barakar Formation of the Satpura Gondwana basin indicating marine influence during sedimentation in a continental interior deltaic setting and suggesting that the basin was connected to oceanic waters during Barakar sedimentation.
In recent years, tide- and wave-influenced deposits that demonstrate marine influence in intracontinental settings have been recognized in association with several coal-bearing successions . Tidal influence in ancient continental deposits has been recognized mostly from estuarine successions deposited in incised valleys overlying sequence boundaries . Discriminating between deposits of tidally influenced deltas and tide-dominated coastal mud flats in the rock record is often difficult. Kvale & Mastalerz have interpreted the tidal deposits associated with the Pennsylvanian sequences of the USA as products of a prograding, coastal tidal flat and associated tidal channels seaward of a coal-forming mire. Unambiguous tidal deposits associated with tidally influenced deltas are rarely documented from intracontinental basins
Identification of tidal signatures within a continental succession has manifold significance: (1) tidal deposits are unambiguous evidence of marine influence;
(2) their recognition allows reconstruction of the broader palaeogeography of the basin, revealing the transition between alluvial and marine domains (Dalrymple et al. 1992; Shanley et al. 1992; Kvale & Barnhill 1994; Brettle et al. 2001), as well as estimation of the inland extent of the tidal influence in ancient settings;
(3) it facilitates chronostratigraphic correlation between marine and alluvial strata, leading to an improved sequence stratigraphic interpretation of the continental succession .
Sedimentation rate analyzer - Industry Spotlight
The ESR-Auto Plus automated analyzer is designed to accurately and precisely measure the sedimentation rate of erythocytes in ESR-vacuum tubes. Capable of testing 10 tubes simultaneously and up to 20 samples per hour, the system can be interfaced with an LIS and offers an automated QC file, built-in printer, and optional barcode scanner.
Organic Valley distribution center to boost economy of southwest Wisc
After more than a year of planning and construction, Organic Valley employees, state and local officials, business leaders and community members gathered in Cashton, Wisc., July 27 to celebrate the grand-opening of a new, $17.5 million Organic Valley Distribution Center. The new facility will serve as the primary warehouse and distribution center for Organic Valley, America’s largest cooperative of organic farmers and one of the nation’s leading organic brands.
The new facility provides ample evidence that organic products have moved from a niche market into mainstream consumption, co-op officials say.
The 80,000-square-foot distribution center is located on 40 acres in the Cashton Greens Business Park, an innovative new development where businesses will create and use renewable energy, such as biomass conversion of manure and sawdust, biodiesel and wind energy. It includes an automated storage and retrieval system.
“The new Organic Valley Distribution Center is a symbol of our co-op’s growth and its ongoing commitment to creating sustainable communities through organic farming,” said co-op CEO George Siemon. “Anchoring our distribution in Cashton is a step to fulfill our mission to help bring economic vitality to rural Wisconsin.”
The facility was built using a number of “green” building practices, including fly ash in the cement, recycled cotton for insulation in the walls and recycled steel throughout the building; a white roof to reflect the sun’s heat and decrease energy costs in refrigeration; waterless urinals; and automatic faucets that recharge their batteries with the flow of water.
Systematic Colocalization Errors between Acridine Orange and EGFP in Astrocyte Vesicular Organelles
Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reliably. We used evanescent-field imaging with spectral fluorescence detection as well as fluorescence lifetime imaging microscopy to demonstrate that green fluorescent AO monomers inevitably coexist with red fluorescing AO dimers, at the level of single astroglial vesicles. The green monomer emission spectrally overlaps with that of EGFP and produces a false apparent colocalization on dual-color images. On fluorophore abundance maps calculated from spectrally resolved and unmixed single-vesicle spectral image stacks, EGFP is obscured by the strong green monomer fluorescence, precluding the detection of EGFP. Hence, extreme caution is required when deriving quantitative colocalization information from images of dim fluorescing EGFP-tagged organelles colabeled with bright and broadly emitting dyes like AO. We finally introduce FM4-64/EGFP dual-color imaging as a remedy for imaging a distinct population of astroglial fusion-competent secretory vesicles.
Fluorescence colocalization imaging is a powerful method for exploring the targeting of molecules to intracellular compartments and to screen for their association and interaction. In such experiments, distinct fluorophores are attached to the molecular targets of interest and imaged into spectrally separated detection channels. The fluorescence intensity in each channel ideally is dominated by spatial and concentration information derived from one fluorophore only. In this case, the dual-color fluorescence intensity images then correspond to diffraction-limited fluorophore maps, which can be overlaid or displayed side-by-side and the amount of colocalization can be calculated using different estimators (1-3).
One recent application of dual-color fluorescence detection has been the identification of vesicular compartments and study of their dynamics and fusion in nonsecretory exocytoses, the primary function of which is the transfer of the organelle membrane and its embedded proteins to the cell surface rather than the release of the vesicular cargo (4), or for studying regulated secretion of vesicular compartments in nonspecialized secretory cells (5), like macrophages, tibroblasts (6) or astrocytes (7) (see, e.g., (8) for review). In contrast to presynaptic nerve terminals or neuroendocrine cells that harbor large numbers of secretory vesicles close to the plasma membrane in readiness for exocytosis, nonsecretory cells lack such obvious morphological and functional specializations. Any attempt to study the release of biologically active substances from these cells must therefore first pinpoint the right vesicular compartment among the numerous endosomes, lysosomes, caveolae, and transport carriers, which is typically done by overexpressing a suitable marker (9) and then specifically follow the fate of the identified organelle, typically by using a second fluorescent reporter of membrane fusion.
Acridine orange (AO) is a weak base and metachromatic, fluorescent cationic dye (10,11) and photosensitizer (12,13) that stains live (12,14) and fixed tissue (15,16) with variable hues of fluorescence. At the cellular level, AO relocation and color change has been widely used as a test for cell viability (17,18), and for studying pH gradients across vesicular (19-22), as well as lyso- (23-25) and endosomal (26,27) membranes.
Dilute AO solutions or AO molecules bound to polyanions and isolated from each other emit green orthochromic fluorescence. Concentrated solutions, aggregates of AO, or molecules bound close by to neighboring sites, have orange or red metachromatic fluorescence (12,28). Note that “metachromasy” is here operationally defined as the hypsochromic (shift in absorption to shorter wavelength) and hypochromic (decrease in intensity of emitted fluorescence) change in color exhibited by certain dyes in the presence of water under the following conditions: 1), increase in dye concentration; 2), temperature decrease; 3), salting out; and 4), interaction with substrates that favor water intercalation and/or proximity or stacking of dye monomers. Like thionine, methylene blue, toluidine blue, rhodamine 6G, and phronine G, AO derives its melachromatic properties from the progressive formation of dimers and higher aggregates with increasing concentration (10,11). Low temperature and ionic strength favor AO metachromasy (27,29,30). Due to these photochemical properties and because of its passive accumulation inside acidic vesicles as well as the pronounced and rapid fluorescence change associated with exocytosis, AO has been popular as a reporter of membrane fusion and vesicle release (20,31,32). The opening of the fusion pore equilibrates the acidic luminal pH, leading to the rapid dequenching of AO fluorescence, which is observed as a loss of metachromatic red fluorescence and a concomitant flash of orthochromatic green fluorescence, followed by the dissipation of AO monomers in the extracellular space.
Modified interactions between salamander life stages caused by wildfire-induced sedimentation
Ecological communities are commonly affected by natural and periodic disturbance events. These disturbances may kill organisms, change competitive outcomes, alter habitats, or introduce new species to existing communities.If a disturbance suddenly adds a new species to a community it may be possible to directly observe the effects of this new species on the interactions of existing species, particularly if the dynamics between existing species had been observed before the introduction.
The effects of wildfire as a disturbance on chaparral plant communities are well studied.Wildfires also impact animal communities but studies examining animal-fire interactions generally consider only short-term population changes in small mammals and birds.Despite studies on the physical changes to stream habitats following fire,little is known about the effects of wildfire on aquatic organisms.Wildfire may indirectly affect aquatic systems by altering the surrounding terrestrial habitat. Fires may cause loss of vegetation, producing soil instability and landslides that change stream geomorphology.After rains, suspended sediments are often higher in streams flowing through burned areas.If organisms are introduced into the stream as a result of sedimentation, interactions among members of the stream community could be modified.
We have taken advantage of a major chaparral wildfire to examine if fire indirectly impacts the feeding ecology of the California newt.Adults breed in perennial streams that flow through the chaparral communities of the Santa Monica Mountains of southern California. Adult T. torosa spend most of the year on land and return to the streams to breed each spring.
Serum sickness-like reaction to cefuroxime: a case report and review of the literature
We report a case of a 34-year-old woman who received cefuroxime, a second-generation cephalosporin, as treatment for mastitis. She subsequently developed a serum sickness-like reaction (SSLR) with a generalized pruritic rash, joint pains, and fever. She improved upon treatment with systemic steroids. SSLR is well-described to cefaclor, a second-generation cephalosporin. However, there is a paucity of reports of SSLR to other cephalosporins such as this case.
A 34-year-old Caucasian woman presented to the dermatology clinic with a 2-week history of mastitis. The patient was breast-feeding following delivery of her sixth child 7 weeks earlier. She had no other significant past medical history. She was treated with 500 mg of oral cefuroxime twice daily and had completed the 10-day prescribed course. Six days into the course of treatment, she developed a rash on the scalp, which generalized over a 5-day time span to involve extensive areas of the trunk, arms, legs, and face. The rash was very pruritic, and individual lesions, which lasted less than 24 hours, were migratory. The patient also complained of intermittent fevers to 101[degrees]F and joint pains of the elbows, knees, and smaller joints of the hands. She specifically denied wheezing or shortness of breath. Her other current medications included prenatal vitamins. At the time of her delivery, 7 weeks previous, she had received pain medications, but no antibiotics. Acetaminophen and diphenhydramine, which she had tolerated well in the past, were taken orally as needed for the rash. Her reported allergies included tetracycline, penicillin, and erythromycin, all of which had caused skin rashes. As the rash continued to worsen in extent and severity, even after the antibiotic was completed, she was treated with oral methylprednisolone for 3 days, then one dose of oral prednisone 30 mg without improvement. She was then referred to the dermatology clinic.
The patient was a thin woman who scratched frequently, but otherwise was in no apparent distress. Skin examination revealed multiple confluent and extensive blanching urticarial plaques with dermatographism on the arms, legs, trunk, and face. No mucosal erosions were present, and there was no edema of the face or extremities. No cervical, occipital, axillary, or inguinal lymphadenopathy was noted. Pulmonary and cardiac examination was unremarkable. She had no swelling or erythema of the joints. The elbows, knees, and joints of the hands exhibited full range of motion. Laboratory studies, including a complete blood count, comprehensive metabolic panel, urinalysis, erythrocyte sedimentation rate, and C3, C4, and CH50 were all within the normal limits.
She was diagnosed with a serum sickness-like reaction (SSLR) to cefuroxime and admitted for 100 mg of intravenous methylprednisolone daily and oral antihistamines. Her symptoms and rash improved within 24 hours, and she was discharged on an extended tapering dose of prednisone starting at 40 mg daily, in addition to oral hydroxyzine, oral doxepin, and topical emollients. She experienced slow but steady resolution of her symptoms over the following 4 weeks.
SSLR is a specific type of drug reaction so named because of its clinical similarity to serum sickness. True serum sickness is a type III hypersensitivity reaction in which clinical signs and symptoms result from deposition of immune complexes in the skin, joints, and other organ systems. In contrast, SSLR is not associated with demonstrable circulating immune complexes. The reaction is acute, self-limited, and has been described in association with a variety of different medications. Although many drugs have been reported to cause SSLR, antibiotics are the major group of offending agents, particularly beta-lactam and sulfonamide antibiotics.
In most cases, signs and symptoms appear about a week after initiation of therapy. The most frequent finding is cutaneous involvement, typically in the form of erythema and urticarial lesions that are often migratory. In the series reported by Hebert et al, many of the urticarial wheals had dusky to purple centers, which were morphologically suggestive of erythema multiforme
The other primary clinical feature is joint involvement. Pain and swelling of the joints is a typical finding, and is usually polyarticular. Affected joints commonly include wrists, ankles, hips, and knees, which can become so severe that patients are unable to walk.
Although fever may occur, other systemic findings are less common. In contrast to true serum sickness, renal and hepatic involvement is rare.
Despite the impressive cutaneous findings and joint symptoms, a hallmark of SSLR is the benign outcome, although some patients do require hospitalization due to their severe symptoms. Treatment is typically symptomatic, usually with antihistamines and analgesics. The use of systemic corticosteroid treatment has been described in retrospective medical record reviews and in a number of case reports, although there is no accepted standardized therapy. Reports of SSLR describe benign outcomes with no sequelae.