Optimizing reduction of western blotting analytical variations: Use of replicate test samples, multiple normalization methods, and sample loading positions.

Western blot (WB) analysis is widely used, but obtaining consistent results can be problematic, especially when using multiple gels. This study examines WB performance by explicitly applying a method commonly used to test analytical instrumentation. Test samples were lysates from RAW 264.7 murine macrophages treated with LPS to activate MAPK and NF-kB signaling targets. Samples from the pooled cell lysates placed in every lane on multiple gels were analyzed by WBs for levels of p-ERK, ERK, IkBß and non-target protein. Different normalization methods and sample groupings were applied to the density values and the resulting coefficients of variation (CV) and ratios of maximal to minimal values (Max/Min) were compared. Ideally with identical sample replicates the CVs would be 0 and the Max/Min 1; deviation indicating introduction of variability by the WB process. Common normalizations to reduce analytical variance, total lane protein, % Control, and p-ERK/ERK ratios, did not have the lowest CVs or Max/Min values. Normalization using the sum of target protein values combined with analytical replication most effectively reduced variability, resulting CV and Max/Min values as low as 5-10% and 1.1. These methods should allow reliable interpretation of complex experiments that require samples to be placed on multiple gels.

Celebrating 60 Years of Accomplishments of the Armed Forces Radiobiology Research Institute1.

Chartered by the U.S. Congress in 1961, the Armed Forces Radiobiology Research Institute (AFRRI) is a Joint Department of Defense (DoD) entity with the mission of carrying out the Medical Radiological Defense Research Program in support of our military forces around the globe. In the last 60 years, the investigators at AFRRI have conducted exploratory and developmental research with broad application to the field of radiation sciences. As the only DoD facility dedicated to radiation research, AFRRI's Medical Radiobiology Advisory Team provides deployable medical and radiobiological subject matter expertise, advising commanders in the response to a U.S. nuclear weapon incident and other nuclear or radiological material incidents. AFRRI received the DoD Joint Meritorious Unit Award on February 17, 2004, for its exceptionally meritorious achievements from September 11, 2001 to June 20, 2003, in response to acts of terrorism and nuclear/radiological threats at home and abroad. In August 2009, the American Nuclear Society designated the institute a nuclear historic landmark as the U.S.'s primary source of medical nuclear and radiological research, preparedness and training. Since then, research has continued, and core areas of study include prevention, assessment and treatment of radiological injuries that may occur from exposure to a wide range of doses (low to high). AFRRI collaborates with other government entities, academic institutions, civilian laboratories and other countries to research the biological effects of ionizing radiation. Notable early research contributions were the establishment of dose limits for major acute radiation syndromes in primates, applicable to human exposures, followed by the subsequent evolution of radiobiology concepts, particularly the importance of immune collapse and combined injury. In this century, the program has been essential in the development and validation of prophylactic and therapeutic drugs, such as Amifostine, Neupogen®, Neulasta®, Nplate® and Leukine®, all of which are used to prevent and treat radiation injuries. Moreover, AFRRI has helped develop rapid, high-precision, biodosimetry tools ranging from novel assays to software decision support. New drug candidates and biological dose assessment technologies are currently being developed. Such efforts are supported by unique and unmatched radiation sources and generators that allow for comprehensive analyses across the various types and qualities of radiation. These include but are not limited to both 60Co facilities, a TRIGA® reactor providing variable mixed neutron and γ-ray fields, a clinical linear accelerator, and a small animal radiation research platform with low-energy photons. There are five major research areas at AFRRI that encompass the prevention, assessment and treatment of injuries resulting from the effects of ionizing radiation: 1. biodosimetry; 2. low-level and low-dose-rate radiation; 3. internal contamination and metal toxicity; 4. radiation combined injury; and 5. radiation medical countermeasures. These research areas are bolstered by an educational component to broadcast and increase awareness of the medical effects of ionizing radiation, in the mass-casualty scenario after a nuclear detonation or radiological accidents. This work provides a description of the military medical operations as well as the radiation facilities and capabilities present at AFRRI, followed by a review and discussion of each of the research areas.

Measuring type I interferon using reporter gene assays based on readily available cell lines.

Cell lines stably transfected with genes responding to Type I interferons (IFN) are potentially a useful alternative to enzyme linked immuo-assays (ELISAs) or assays based on resistance of a test cell line to virus infection using cell death or infection endpoints. Increasingly available are a variety of commercial cell lines developed for reporter gene assays (RGAs) which are responsive to IFN exposure. These cells produce a soluble gene product which can be readily quantified using multiwell plate spectrophotometers or luminometers. We have investigated RAW-Blue ISG™ and B16-Blue IFNα/ß™ cells (InvivoGen) which produce secreted embryonic alkaline phosphatase (SEAP) as a RGA to measure Interferon alpha (IFNα) and beta (IFNß). These cells showed a log-linear response over 4 logs of IFN concentration between 10 and 100,000 Units/ml (U/ml). Concentration dependent responses could be observed as early as 6 h but greater sensitivity was obtained at 24 h. Neutralizing antibodies to IFNα and IFNß reduced the response to baseline. As proof of principle supernatants from RAW 264.7 (murine macrophage; parental cell line) infected with 1 multiplicity of infection (moi) of influenza A virus (X31/H3N2) were used as test samples. Pre-treatment of the supernatant with anti-IFNα failed to reduce the cell response but it was reduced to background by anti-IFNß. The high level of IFNß but very low level of IFNα was confirmed by ELISA. Availability, ease of use and maintenance, and possible cost savings make application of this reporter gene cell approach a valuable alternative to other methods for measuring Type I interferon.

Reduced engagement with social stimuli in 6-month-old infants with later autism spectrum disorder: a longitudinal prospective study of infants at high familial risk.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects more than 1 % of the population and close to 20 % of prospectively studied infants with an older sibling with ASD. Although significant progress has been made in characterizing the emergence of behavioral symptoms of ASD, far less is known about the underlying disruptions to early learning. Recent models suggest that core aspects of the causal path to ASD may only be apparent in early infancy. Here, we investigated social attention in 6- and 12-month-old infants who did and did not meet criteria for ASD at 24 months using both cognitive and electrophysiological methods. We hypothesized that a reduction in attention engagement to faces would be associated with later ASD. METHODS: In a prospective longitudinal design, we used measures of both visual attention (habituation) and brain function (event-related potentials to faces and objects) at 6 and 12 months and investigated the relationship to ASD outcome at 24 months. RESULTS: High-risk infants who met criteria for ASD at 24 months showed shorter epochs of visual attention, faster but less prolonged neural activation to faces, and delayed sensitization responses (increases in looking) to faces at 6 months; these differences were less apparent at 12 months. These findings are consistent with disrupted engagement of sustained attention to social stimuli. CONCLUSIONS: These findings suggest that there may be fundamental early disruptions to attention engagement that may have cascading consequences for later social functioning.

Early activation of MAP kinases by influenza A virus X-31 in murine macrophage cell lines.

Early molecular responses to Influenza A (FLUA) virus strain A/X-31 H3N2 in macrophages were explored using J774.A1 and RAW 264.7 murine cell lines. NF-kappa B (NFκB) was reported to be central to FLUA host-response in other cell types. Our data showed that FLUA activation of the classical NFκB dependent pathway in these macrophages was minimal. Regulator proteins, IkappaB-alpha and -beta (IκBα, IκBß), showed limited degradation peaking at 2 h post FLUA exposure and p65 was not observed to translocate from the cytoplasm to the nucleus. Additionally, the non-canonical NFκB pathway was not activated in response to FLUA. The cells did display early increases in TNFα and other inflammatory cytokine and chemokine production. Mitogen activated phosphokinase (MAPK) signaling pathways are also reported to control production of inflammatory cytokines in response to FLUA. The activation of the MAPKs, cJun kinases 1 and 2 (JNK 1/2), extracellular regulated kinases 1 and 2 (ERK 1/2), and p38 were investigated in both cell lines between 0.25 and 3 h post-infection. Each of these kinases showed increased phosphorylation post FLUA exposure. JNK phosphorylation occurred early while p38 phosphorylation appeared later. Phosphorylation of ERK 1/2 occurred earlier in J774.A1 cells compared to RAW 264.7 cells. Inhibition of MAPK activation resulted in decreased production of most FLUA responsive cytokines and chemokines in these cells. The results suggest that in these monocytic cells the MAPK pathways are important in the early response to FLUA.

A multidisciplinary approach to teach responses to weapons of mass destruction and terrorism using combined simulation modalities.

STUDY OBJECTIVE: To reinforce concepts presented in the lectures; understand the complexity and speed of casualty and information generation during a Weapons of Mass Destruction and Terrorism (WMD/T) event; experience the novelty of combined weapons' effects; recognize the time course of the various chemical, biological, and radiation agents; and make challenging decisions with incomplete and conflicting information. SETTINGS: Two environments simulated simultaneously: one a major trauma center emergency room (ER) with two patient simulators and several human actors; the other an Emergency Operations Command Center (EOC). TARGET AUDIENCE: Students for this course included: clinicians, scientists, military and intelligence officers, lawyers, administrators, and logistic personnel whose jobs involve planning and executing emergency response plans to WMD/T. SIMULATION SCRIPT: A WMD/T attack in Washington, D.C., has occurred. Clinical students performed in their real life roles in the simulated ER, while nonclinical students did the same in the simulated EOC. Six ER casualties with combined WMD/T injuries were presented and treated over 40 minutes. In the EOC, each person was given his or her role title with identification tag. The EOC scenario took cues from the action in the ER via two television (TV) news feeds and telephone calls from other Emergency Operations Assets. PERFORMANCE EXPECTATIONS: Students were expected to actively engage in their roles. Student performances were self-evaluated during the debriefing. DEBRIEFING: The two groups were reunited and debriefed utilizing disaster crisis resource management tools. ASSESSMENT OF EFFECTIVENESS: Students answered an 18-point questionnaire to help evaluate the usefulness and acceptance of multimodality patient simulation. LESSONS LEARNED: Large-scale multimodality patient simulation can be used to train both clinicians and nonclinicians for future events of WMD/T. Students accepted the simulation experience and thought that scenario was appropriately realistic, complex, and overwhelming. Difficulties include the extensive man-hours involved in designing and presenting the live simulations. EOC-only sessions could be staged with only a few video cassette recorders, TVs, telephones, and callers.

A Multidisciplinary Approach to Teach Responses to Weapons of Mass Destruction and Terrorism Using Combined Simulation Modalities.

STUDY OBJECTIVE: To reinforce concepts presented in the lectures; understand the complexity and speed of casualty and information generation during a Weapons of Mass Destruction and Terrorism (WMD/T) event; experience the novelty of combined weapons' effects; recognize the time course of the various chemical, biological, and radiation agents; and make challenging decisions with incomplete and conflicting information. SETTINGS: Two environments simulated simultaneously: one a major trauma center emergency room (ER) with two patient simulators and several human actors; the other an Emergency Operations Command Center (EOC). TARGET AUDIENCE: Students for this course included: clinicians, scientists, military and intelligence officers, lawyers, administrators, and logistic personnel whose jobs involve planning and executing emergency response plans to WMD/T. SIMULATION SCRIPT: A WMD/T attack in Washington, D.C., has occurred. Clinical students performed in their real life roles in the simulated ER, while nonclinical students did the same in the simulated EOC. Six ER casualties with combined WMD/T injuries were presented and treated over 40 minutes. In the EOC, each person was given his or her role title with identification tag. The EOC scenario took cues from the action in the ER via two television (TV) news feeds and telephone calls from other Emergency Operations Assets. PERFORMANCE EXPECTATIONS: Students were expected to actively engage in their roles. Student performances were self-evaluated during the debriefing. DEBRIEFING: The two groups were reunited and debriefed utilizing disaster crisis resource management tools. ASSESSMENT OF EFFECTIVENESS: Students answered an 18-point questionnaire to help evaluate the usefulness and acceptance of multimodality patient simulation. LESSONS LEARNED: Large-scale multimodality patient simulation can be used to train both clinicians and nonclinicians for future events of WMD/T. Students accepted the simulation experience and thought that scenario was appropriately realistic, complex, and overwhelming. Difficulties include the extensive man-hours involved in designing and presenting the live simulations. EOC-only sessions could be staged with only a few video cassette recorders, TVs, telephones, and callers.

Active and inactive influenza virus induction of tumor necrosis factor-alpha and nitric oxide in J774.1 murine macrophages: modulation by interferon-gamma and failure to induce apoptosis.

Infection of J774.1 murine macrophages by influenza A virus (IAV) induces two major responses, production of host defense molecules and death by apoptosis. We investigated whether induction of two cytotoxic compounds, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), directly caused IAV-induced apoptosis, and whether induction could be modulated by interferon-gamma (IFN-gamma) or the replication competence of the virus. Live IAV potently induced production of both TNF-alpha and NO, but UV inactivated virus was a poor inducer of both molecules. When cells were pre-treated with IFN-gamma, inactive IAV became as effective an inducer of NO, but not TNF-alpha, as live IAV. Amantadine, which antagonizes viral entry and replication, partly inhibited TNF-alpha and NO production in unprimed cells, but did not inhibit NO in IFN-gamma primed cells. IAV-induced cytotoxicity was not due to the induction of TNF-alpha or NO. Cells were insensitive to either TNF-alpha-containing supernatants or to recombinant TNF-alpha. Anti-TNF-alpha antibody did not protect cells from IAV-induced cell death, and anti-oxidants that inhibited TNF-alpha production also failed to increase cell survival. Inhibitors of NO production did not protect from IAV-induced cell death, either alone or in combination with superoxide dismutase (SOD). We conclude that, even though IAV was a potent inducer of TNF-alpha and NO in macrophages, IAV-induced apoptosis was not mediated directly by them. Importantly, viral replication was not required for the induction of TNF-alpha or NO, and the action of inactive IAV could be potentiated by IFN-gamma.

Influenza virus induction of apoptosis by intrinsic and extrinsic mechanisms.

It is now firmly established that apoptosis is an important mechanism of influenza virus-induced cell death both in vivo and in vitro. Data are predominantly from experiments with influenza A virus and in vitro experimental systems. Multiple influenza virus factors have been identified that can activate intrinsic or extrinsic apoptotic induction pathways. Currently there is no evidence for influenza virus directly accessing the apoptosis execution factors. The best-studied influenza virus inducers of apoptosis are dsRNA, NS1, NA, and a newly described gene product PB1-F2. PB1-F2 is the only influenza virus factor to date identified to act intrinsically by localization and interaction with the mitochondrial-dependent apoptotic pathway. Both dsRNA and NA have been shown to act via an extrinsic mechanism involving proapoptotic host-defense molecules: PKR by induction of Fas-Fas ligand and NA by activation of TGF-beta. PKR is capable of controlling several important cell-signaling pathways and therefore may have multiple effects; a predominant one is increased interferon (IFN) production and activity. NS1 has been shown to be both proapoptotic and antiapoptotic. Use of influenza virus NS1 deletion mutants has provided evidence for NS1 interference with apoptosis, IFN induction, and related cell-signaling pathways. Influenza virus also has important exocrine paracrine effects, which are likely mediated via TNF family ligands and oxygen, free radicals capable of inducing apoptosis. Little is known about activation of inhibitors of apoptosis such as inhibitory apoptotic proteins. Whether all these factors always have a role in influenza virus-induced apoptosis is unknown. The kinetics of synthesis of influenza virus factors affecting apoptosis during the replication cycle may be an important aspect of apoptosis induction.

Comparison of gamma and neutron radiation inactivation of influenza A virus.

Radiation inactivation of viral pathogens has potential application in sterilization and in the manufacture of biological reagents, including the production of non-infectious viral antigens. Viral inactivation by gamma radiation has been extensively investigated, but few direct comparisons to other qualities of radiation have been explored. Experiments were designed to examine direct radiation damage by both gamma photons (gamma) and neutrons (n) while minimizing methodological differences. Frozen samples of influenza A X31/H3N2 and PR8/H1N1 were exposed to gamma and n at doses between 0 and 15.6 kGy. Other experimental parameters, including dose-rate, were not varied. Virus titers were determined by tissue culture infectious dose (TCID(50)) and plaque forming unit (PFU) assays. D(10) values, kGy per log reduction, were calculated from these assays. PR8 D(10) values based on PFU assays were approximately 2 and 5 kGy for gamma and n exposures, respectively, and those based on TCID(50) were approximately 6 and 14 kGy. Similar results were obtained for the A/X31 strain. The data demonstrate that gamma was 2-3-fold more effective than n, with a relative biological effectiveness (RBE) range of 0.43-0.65. These neutron results are likely the first reported for a medically relevant virus. PAGE analysis of viral proteins and RNAs failed to show macromolecular damage. D(10) values were found to be similar to a broad summary of previously reported gamma inactivation values for other virus types. The dependence of the magnitudes of D(10) on titer assay in this study suggests that more than one titer method should be used to determine if complete inactivation has occurred.

Improvements in methods for calculating virus titer estimates from TCID50 and plaque assays.

Calculation of titer estimates and use of titer reduction assays are fundamental approaches used by virologists. Titer assays being biological assays and based on limiting dilution methods require good error control, both methodologically and analytically. The need for good statistical analysis is likely to become even greater as in clinical, manufacturing, as well as the research settings, improved analytical criteria, quality control, and assurance standards are adopted. Furthermore, increasingly, virus titer assays are based on high throughput methods, which generate continuous rather than traditional quantal data. Described here are two different weighted linear regression methods to determine TCID50 and PFU titers from CPE assays. The TCID50 analysis makes use of a generalized least squares approach using continuous colorimetric data. The plaque analysis makes use of weighted least squares forced through the origin using quantal plaque data generated by serial dilutions. Both methods are improvements in titer and error estimation compared to simpler calculation methods. These methods may have greatest value when lack of experimental material or costs of analysis precludes extensive replicate titer determinations but good estimates of titers and/or treatment differences are essential.

Characterization of influenza virus-induced death of J774.1 macrophages.

The mechanism and role of influenza virus (IV)-induced pathogenesis of macrophages during respiratory infection are ill defined. Reported here are findings on IV-induced cytopathic effects (CPEs) for an in vitro experimental system using the murine macrophage cell line J774.1. CPE was elicited by 0.2 or greater multiplicity of infection (m.o.i.). CPEs showed a lag of 6-8 h postinfection and occurred most rapidly between 6 and 12 h. J774.1 cells did not support productive IV replication, but immunofluorescence demonstrated that IV protein synthesis occurred. Light microscopy and DNA staining showed that after death cells had very condensed cytoplasm and nuclei. Cell remnants were surrounded by intact plasma membrane (PM) as demonstrated by exclusion of a membrane-impermeant dye. Time-lapse video microscopy recordings between 6 and 10 h postinfection showed sequential structural changes, including previously undescribed events. Notable changes were a rapid cytokinesis (zeiosis; "cell boiling"), followed by nuclear shrinkage, and an unusual transient blebbing of the PM. DNA fragmentation occurred after 12 h, producing a wide size range. UV-inactivated virus failed to induce CPEs, and CPE was blocked by amantadine. N-Acetylcysteine and pyrrolidine dithiocarbamate, but not other inhibitors of reactive oxygen intermediates, reduced or blocked the CPE. Most changes observed are those attributed to apoptotic processes rather than necrotic cell death. The kinetics and inhibitor effects suggest that IV infection and replication must be initiated to activate CPEs.

Teenage childbearing. An adaptive strategy for the socioeconomically disadvantaged or a strategy for adapting to socioeconomic disadvantage?

OBJECTIVE: To examine the relation between childbearing and educational and vocational achievements of American females high school students. DATA SOURCE: Articles published in English during the past decade about the educational, vocational, and socioeconomic sequelae of childbearing among female high school students. DATA SELECTION: Articles that did not contain data about the relation between adolescent childbearing and educational and vocational achievement were excluded. DATA SYNTHESIS: Most females who begin childbearing during adolescence obtain less schooling and poorer-paying jobs than do females who postpone childbearing. The reasons for this are elusive. Differences in the family and cultural backgrounds of early (high school-age) and later (18 years and older) childbearers explain some but not all of the association between early childbearing and educational and vocational underachievement. The effect of childbearing preferences on the educational and vocational achievements of teenagers has not been studied adequately. Lack of concrete information could result in underestimation of the effect of early childbearing on the socioeconomic well-being of young Americans, and create the impression that adolescent pregnancy is an adaptive response to urban poverty. CONCLUSIONS: As much as the long-term socioeconomic sequelae of adolescent childbearing reflect factors that influence the judgments young people make about the costs and benefits of contraception and parenthood, adolescent childbearing is a means of adapting to urban poverty. Thus postponing adolescent conceptions and parenthood may have a less important effect on the socioeconomic well-being of young Americans than expected.

Evaluation of triple-band filters for quantitative epifluorescence microscopy.

The performance characteristics of two sets of triple-band epifluorescence filters have been evaluated for use with digitally enhanced fluorescence microscopy. Use of such filters, at most, requires movement of the excitation filter, while the dichroic and emission filters remain fixed, allowing multi-wavelength imaging to be performed on standard microscopes. The dyes appropriate for use with these particular filters include Texas Red (TR), Bodipy (BD), FITC and Cascade Blue (CB), four fluorophores now commonly conjugated to both immunochemical probes and other proteins and lipids of biological interest. Good spectral separation existed for most experimental conditions allowing accurate localization of the different fluorophores during multi-wavelength imaging. Anomalous responses were observed during near-UV excitation at high concentration for some dyes. Scanning spectrofluorometry demonstrated that concentration-dependent spectral shifts occurred, resulting in large increases in near-UV absorbance. Despite the complexity of concentration and dye-interaction effects, quantitative measurements of dye concentration could be made, even in regions of multiple dye co-localization. Therefore, multi-band pass filters are an additional valuable approach for performing quantitative fluorescence microscopic imaging.

Differences in dispersion of influenza virus lipids and proteins during fusion.

Digitally enhanced low-light-level fluorescence video microscopy and immunochemical staining were used to examine influenza virus envelope lipid and protein redistribution during pH-induced fusion. Video microscopy was performed using viruses labeled with either the lipid analogue octadecylrhodamine B (R18) or fluorescein isothiocyanate (FITC) covalently linked to envelope proteins. Viruses were bound to human red blood cells, and the pattern and intensity of fluorescence were monitored for 30 min while cell-virus complexes were perfused with pH 7.4 or 4.8 media at temperatures either above or below 20 degrees C. R18 showed complete redistribution and dequenching by 30 min at all incubation temperatures, confirming reports that viral fusion occurs at subphysiological temperatures. FITC-labeled protein showed spatial redistribution at 28 degrees C but no change at low temperature. Electron microscopy observations of immunochemical staining of viral proteins confirmed both that protein redistribution at 37 degrees C was slower than R18 and the failure of movement within 30 min at 16 degrees C. Video microscopy monitoring of RNA staining by acridine orange of virus-cell complexes showed redistribution to the RBCs at all temperatures but only after low pH-induced fusion. The results are consistent with differential dispersion of viral components into the RBC and the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation.

Transient domains induced by influenza haemagglutinin during membrane fusion.

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy.

MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns. A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450-500 nm excitation/greater than 510 nm emission) or rhodamine (514 nm excitation/greater than 530 emission) type dyes. A second 5- to 10-fold brighter emission for 450-500 nm excitation was observed, which was unusual in that each cell appeared to be outlined. Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled "domes" and was the source of this fluorescent emission. Quantitative measurements showed that exposure to cultures to 10 microM riboflavin resulted in accumulation in domes of 565 +/- 80 microM. The transport rate was calculated to be 189 +/- 30 pmol/min-cm2. One mM probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10 mM nearly abolished the uptake. The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatible with low-light level imaging. Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process.

Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.

We have used intensified video fluorescence microscopy and digital image processing to observe and quantitate influenza virus (A/PR8/34/H1N1) fusion to human erythrocyte membranes. Viruses labeled with the lipid probe octadecylrhodamine B (R18) were seen to undergo fluorescence dequenching and eventual disappearance after exposure to pH levels known to induce virus-cell membrane fusion. Quantitative intensity measurements of single individual particles were possible. From these fluorescence data it has been possible to calculate the fraction of R18 dye molecules transferred from the virus to the cell. The redistribution of the lipid probe upon fusion at pH 5.0 had a t1/2 of 46 s, longer than expected for a free-diffusion model. The R18 loss was approximately twice as fast at pH 5.0 as at pH 5.1. No obvious delay until the start of fluorescence dequenching was observed after the pH changes, suggesting that activation processes are faster than the time resolution, 1-5 s, of the current method.

Two K+ channel types, muscarinic agonist-activated and inwardly rectifying, in a Cl- secretory epithelium: the avian salt gland.

Patches of membrane on cells isolated from the nasal salt gland of the domestic duck typically contained two types of K+ channel. One was a large-conductance ("maxi") K+ channel which was activated by intracellular calcium and/or depolarizing membrane voltages, and the other was a smaller-conductance K+ channel which exhibited at least two conductance levels and displayed pronounced inward rectification. Barium blocked both channels, but tetraethylammonium chloride and quinidine selectively blocked the larger K+ channel. The large K+ channel did not appear to open under resting conditions but could be activated by application of the muscarinic agonist, carbachol. The smaller channels were open under resting conditions but the gating was not affected by carbachol. Both of these channels reside in the basolateral membranes of the Cl- secretory cells but they appear to play different roles in the life of the cell.