The relationship between pyramidal cell morphology and efferent target was investigated in layer 6 of cat primary visual cortex (area 17). Layer 6 has 2 projections, one to the lateral geniculate nucleus (LGN) and another to the visual claustrum. The cells of origin of each projection were identified by retrograde transport of fluorescent latex microspheres. The labeled cells were visualized in brain slices prepared from area 17, using an epifluorescence compound microscope modified for intracellular recording. Individual retrogradely labeled cells were penetrated and intracellularly stained with Lucifer yellow to visualize the patterns of axons and dendrites associated with each projection. The neurons that give rise to the 2 projections had very different patterns of dendrites and local axonal collaterals, but the patterns within each group were highly stereotyped. The differences between their axonal collaterals were particularly dramatic. Claustrum projecting cells had fine, horizontally directed collaterals that arborized exclusively in layer 6 and lower layer 5. Most LGN projecting cells had virtually no horizontal arborization in layer 6. Instead, they sent widespread collaterals vertically, which arborized extensively in layer 4. The apical dendrites of the 2 groups also differed markedly. Claustrum projecting cells had apical dendrites reaching to layer 1, with branches in layer 5 only, while LGN projecting cells never had an apical dendrite reaching higher than layer 3, with side branches in layers 5 and 4. Therefore, each efferent target must receive inputs from neurons whose synaptic connections within area 17 are significantly different from those of neurons projecting to other targets. This further suggests that distinct visual response properties should be associated with each projection. In addition to the claustrum and LGN projecting cells, about 20% of layer 6 pyramidal neurons lacked an efferent axon. Morphologically, most resembled LGN projecting neurons, but a few had characteristics of claustrum projecting cells. These neurons may represent cells that either failed to make an efferent connection or cells that lost an efferent axon during development. Their frequency suggests that such intrinsic, presumably excitatory, neurons may play a significant role in cortical processing.

During reproduction in flowering plants, pollen grains form a tube that grows in a polarized fashion through the female tissues to eventually fertilize the egg cell. These highly polarized pollen tubes have a rapid rate of growth that is supported by a tip-focused delivery of membrane and cell wall components. To gain a better understanding of how this growth is regulated, we investigated the function RABA4D, a member of the Arabidopsis thaliana RabA4 subfamily of Rab GTPase proteins. Here, we show that RABA4D was expressed in a pollen-specific manner and that enhanced yellow fluorescent protein (EYFP)-RabA4d-labeled membrane compartments localized to the tips of growing pollen tubes. Mutant pollen in which the RABA4D gene was disrupted displayed bulged pollen tubes with a reduced rate of growth in vitro and displayed altered deposition of some cell wall components. Expression of EYFP-RabA4d restored wild-type phenotypes to the raba4d mutant pollen tubes, while expression of EYFP-RabA4b did not rescue the raba4d phenotype. In vivo, disruption of RABA4D resulted in a male-specific transmission defect with mutant raba4d pollen tubes displaying aberrant growth in the ovary and reduced guidance at the micropyle. We propose that RabA4d plays an important role in the regulation of pollen tube tip growth.

Why the leaves of many woody species accumulate anthocyanins prior to being shed has long puzzled biologists because it is unclear what effects anthocyanins may have on leaf function. Here, we provide evidence for red-osier dogwood (Cornus stolonifera) that anthocyanins form a pigment layer in the palisade mesophyll layer that decreases light capture by chloroplasts. Measurements of leaf absorbance demonstrated that red-senescing leaves absorbed more light of blue-green to orange wavelengths (495-644 nm) compared with yellow-senescing leaves. Using chlorophyll a fluorescence measurements, we observed that maximum photosystem II (PSII) photon yield of red-senescing leaves recovered from a high-light stress treatment, whereas yellow-senescing leaves failed to recover after 6 h of dark adaptation, which suggests photo-oxidative damage. Because no differences were observed in light response curves of effective PSII photon yield for red- and yellow-senescing leaves, differences between red- and yellow-senescing cannot be explained by differences in the capacities for photochemical and non-photochemical light energy dissipation. A role of anthocyanins as screening pigments was explored further by measuring the responses PSII photon yield to blue light, which is preferentially absorbed by anthocyanins, versus red light, which is poorly absorbed. We found that dark-adapted PSII photon yield of red-senescing leaves recovered rapidly following illumination with blue light. However, red light induced a similar, prolonged decrease in PSII photon yield in both red- and yellow-senescing leaves. We suggest that optical masking of chlorophyll by anthocyanins reduces risk of photo-oxidative damage to leaf cells as they senesce, which otherwise may lower the efficiency of nutrient retrieval from senescing autumn leaves.

More than 1.4 billion people depend on water from the Indus, Ganges, Brahmaputra, Yangtze, and Yellow rivers. Upstream snow and ice reserves of these basins, important in sustaining seasonal water availability, are likely to be affected substantially by climate change, but to what extent is yet unclear. Here, we show that meltwater is extremely important in the Indus basin and important for the Brahmaputra basin, but plays only a modest role for the Ganges, Yangtze, and Yellow rivers. A huge difference also exists between basins in the extent to which climate change is predicted to affect water availability and food security. The Brahmaputra and Indus basins are most susceptible to reductions of flow, threatening the food security of an estimated 60 million people.

To construct chimeric YF/DEN viruses (ChimeriVax-DEN), the premembrane (prM) and envelope (E) genes of yellow fever (YF) 17D virus were replaced with those of each wild-type (WT) dengue (DEN) virus representing serotypes 1 to 4. ChimeriVax-DEN1-4 vaccine viruses were prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA (F. Guirakhoo, J. Arroyo, K. V. Pugachev, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Soike, M. Ratteree, and T. P. Monath, J. Virol. 75:7290-7304, 2001; F. Guirakhoo, K. Pugachev, J. Arroyo, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Draper, and T. P. Monath, Virology 298:146-159, 2002). Progeny viruses were subjected to three rounds of plaque purifications to produce the Pre-Master Seed viruses at passage 7 (P7). Three further passages were carried out using U.S. current Good Manufacturing Practices (cGMP) to produce the Vaccine Lot (P10) viruses. Preclinical studies demonstrated that the vaccine candidates are replication competent and genetically stable and do not become more neurovirulent upon 20 passages in Vero cells. The safety of a tetravalent vaccine was determined and compared to that of YF-VAX in a formal monkey neurovirulence test. Brain lesions produced by the tetravalent ChimeriVax-DEN vaccine were significantly less severe than those observed with YF-VAX. The immunogenicity and protective efficacy of four different tetravalent formulations were evaluated in cynomolgus monkeys following a single-dose subcutaneous vaccination followed by a virulent virus challenge 6 months later. All monkeys developed low levels of viremia postimmunization, and all the monkeys that had received equal concentrations of either a high-dose (5,5,5,5) or a low-dose (3,3,3,3) formulation seroconverted against all four DEN virus serotypes. Twenty-two (92%) of 24 monkeys were protected as determined by lack of viremia post-challenge. This report is the first to demonstrate the safety of a recombinant DEN virus tetravalent vaccine in a formal neurovirulence test, as well as its protective efficacy in a monkey challenge model.

ChimeriVax-JE is a live, attenuated vaccine against Japanese encephalitis, using yellow fever (YF) 17D vaccine as a vector. In a double-blind phase 2 trial, 99 adults received vaccine, placebo, or YF 17D vaccine (YF-VAX). ChimeriVax-JE was well tolerated, with no differences in adverse events between treatment groups. Viremias resulting from administration of ChimeriVax-JE and YF-VAX were of short duration and low titer; 82 (94%) of 87 subjects administered graded doses (1.8-5.8 log(10)) of ChimeriVax-JE developed neutralizing antibodies. A second dose, administered 30 days later, had no booster effect. Previous inoculation with YF did not interfere with ChimeriVax-JE, but there was a suggestion (not statistically significant) that ChimeriVax-JE interfered with YF-VAX administered 30 days later. A separate study explored immunological memory both in subjects who had received ChimeriVax-JE 9 months before and in ChimeriVax-JE-naive subjects challenged with inactivated mouse-brain vaccine (JE-VAX). Anamnestic responses were observed in preimmune individuals. ChimeriVax-JE appears to be a safe vaccine that provides protective levels of neutralizing antibody after a single dose.

Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germline transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species.

The influence of emotional stimuli on source memory was investigated by using emotionally valenced words. The words were colored blue or yellow (Experiment 1) or surrounded by a blue or yellow frame (Experiment 2). Participants were asked to associate the words with the colors. In both experiments, emotionally valenced words elicited enhanced free recall compared with nonvalenced words; however, recognition memory was not affected. Source memory for the associated color was also enhanced for emotional words, suggesting that even memory for contextual information is benefited by emotional stimuli. This effect was not due to the ease of semantic clustering of emotional words because semantically related words were not associated with enhanced source memory, despite enhanced recall (Experiment 3). It is suggested that enhancement resulted from facilitated arousal or attention, which may act to increase organization processes important for source memory.

Mammalian reproduction is dependent upon intermittent delivery of luteinizing hormone-releasing hormone (LHRH) to the anterior pituitary. This mode of secretion is required to sensitize maximally the gonadotrophs to LHRH stimulation and to regulate gonadotropin gene expression. While LHRH secretion is pulsatile in nature, the origin of the pulse generator is unknown. In this report, we show that this oscillator could be located within the LHRH neuronal network. When immortalized LHRH neurons are placed into a perifusion system, LHRH is secreted into the medium in a pulsatile fashion under basal conditions. LHRH secretion and the number of LHRH pulses are reduced when calcium is removed from the medium. Perifusion also influences pro-LHRH processing, since the molar ratio of its processed products varies dramatically when the cells are transferred from a static system. Several different cellular mechanisms may underlie these changes in secretion and processing. Lucifer yellow experiments reveal that some cells are dye-coupled. Hence, these cells could be electrically coupled through gap junctions such that secretion from individual cells could be coordinated. Secretion could also be synchronized through the observed synapse-like contacts. These contacts could perform a negative-feedback role to regulate not only the amount of LHRH released but also the molecular forms secreted. The organization of LHRH neurons into interconnected clusters could serve to coordinate LHRH secretion from individual cells and, thereby, orchestrate functions in vivo as diverse as the onset of puberty, the timing of ovulation, and the duration of lactational infertility.

Ten fluorescent fractions originating from the chloroform extracts of retinal pigment epithelial (RPE) cells of human donor eyes (ages 52-98 yr) have been separated and characterized by UV-vis absorbance and corrected fluorescence spectroscopy. The semipurified fluorophores fall into four categories based upon their spectral properties: green-emitting fluorophores, a golden yellow-emitting fluorophore, yellow-green-emitting fluorophores and orange-red-emitting fluorophores. All share common absorbance peaks around 280- and 330 nm, and the orange-red-emitting fluorophores also exhibit a strong absorbance peak at 420 nm. No significant visible-emitting fluorophores were detected in the methanol-water phase of these extracts. While these fluorophores are abundant in extracts from adult-derived RPE, most of the fluorophores occur in much lower amounts in RPE extracts from human donors under 10 yr of age. Eyes from child human donors also have much less RPE lipofuscin than those from adult donors, suggesting that most of the fluorophores are lipofuscin derived. This interpretation is supported by the previous finding that all of the fluorophores from whole RPE are also present in extracts of purified lipofuscin granules. Characterization of the chromatographic and spectral properties of the chloroform-soluble fluorescent components from the human RPE provides an important tool for determining the mechanism of RPE lipofuscin fluorophore formation. The absorbance properties defined here are of significance to investigations into the photobiology of the RPE and to those using laser therapy in treatment of age-related retinal diseases.

The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.

Variation in human skin/hair pigmentation is due to varied amounts of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eu- and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many mammals. We have sequenced the MC1R gene in 121 individuals sampled from world populations with an emphasis on Asian populations. We found variation at five nonsynonymous sites (resulting in the variants Arg67Gln, Asp84Glu, Val92Met, Arg151Cys, and Arg163Gln), but at only one synonymous site (A942G). Interestingly, the human consensus protein sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency (7%) in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. The MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon was sequenced to study the evolution of MC1R. The ancestral human MC1R sequence is identical to the human consensus protein sequence, while MC1R varies considerably among higher primates. A comparison of the rates of substitution in genes in the melanocortin receptor family indicates that MC1R has evolved the fastest. In addition, the nucleotide diversity at the MC1R locus is shown to be several times higher than the average nucleotide diversity in human populations, possibly due to diversifying selection.

The shoot apex of overwintering perennials ceases its morphogenetic activity at the end of the growing season and transforms into a bud which is dormant and freezing-tolerant. In birch (Betula pubescens) these events are triggered by short photoperiod, and involve the production of 1,3-beta-D-glucan containing sphincters on the plasmodesmata. As a result, all symplasmic pathways shut down. Here we show that breakage of bud dormancy by chilling involves restoration of the symplasmic organization of the meristem. This restoration is likely to be mediated by 1,3-beta-D-glucanase, which was present in small spherosome-like vacuoles that arose de novo during dormancy induction. During chilling these vacuoles were displaced from the bulk cytoplasm to the cortical cytoplasm where they became aligned with the plasma membrane, often associated with plasmodesmata. At this stage the enzyme also appeared outside the vacuoles. During chilling, 1,3-beta-D-glucan disappeared from the plasmodesmal channels and wall sleeves, and the plasmodesmata regained the capacity for cell-cell transport, as demonstrated by microinjection of Lucifer Yellow CH and Fluorescein-tagged gibberellic acid. Collectively, the present experiments demonstrate that restoration of the symplasmic organization of the meristem is indispensable for the release of buds from dormancy and the assumption of a proliferation-competent state, and implicate 1,3-beta-D-glucanase action at the plasmodesmata. Based on these findings we propose a model for 'dormancy cycling' which depicts the meristem as passing through three sequential states of cellular communication with characteristic sensitivities to distinct environmental cues.

Host odours play a major role in the orientation and host location of blood-feeding mosquitoes. Anopheles gambiae Giles sensu stricto, which is the most important malaria vector in Africa, is a highly anthropophilic mosquito species, and the host-seeking behaviour of the females of this mosquito is guided by volatiles of human origin. Ammonia, lactic acid and several carboxylic acids are known to be present in the human odour blend. We investigated the effect of these compounds on naive female mosquitoes using a dual-port olfactometer. Ammonia was an attractant on its own, whereas lactic acid was not attractive. Carboxylic acids, offered as a mixture of 12 compounds, were repellent at the concentration tested. The addition of ammonia to the carboxylic acid mixture overruled the repellent effect of the latter. Combining ammonia with either lactic acid or the carboxylic acids did not enhance the attractiveness of ammonia alone. However, a synergistic effect was found when ammonia, lactic acid and the carboxylic acids were applied as a blend. Our findings indicate that An. gambiae s.s. relies on the combination of ammonia, lactic acid and carboxylic acids in its orientation to human hosts. The role of lactic acid in this tripartite synergism differs from that reported for the yellow fever mosquito Aedes aegypti.

The objective of this study was to investigate how 6 commonly used solvent systems affected the yields of phenolic substances and the antioxidant capacity of extracts from 8 major classes of food legumes. Several antioxidant-related phytochemical compositions, namely, total phenolic content (TPC), total flavonoids content (TFC), and condensed tannins content (CTC), were investigated. In addition, antioxidant activities were tested using 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical scavenging, ferric-reducing antioxidant power (FRAP), and the oxygen radical absorbance capacity (ORAC). The results showed that the 50% acetone extracts exhibited the highest TPC for yellow pea, green pea, chickpea, and yellow soybean. Acidic 70% acetone (+0.5% acetic acid) extracts exhibited the highest TPC, TFC, and FRAP values for black bean, lentil, black soybean, and red kidney bean. The 80% acetone extracts exhibited the highest TFC, CTC, and DPPH-free radical scavenging activity for yellow pea, green pea, chickpea, and yellow soybean. The 70% ethanol extracts exhibited the greatest ORAC value for all selected legumes. These results indicated that solvents with different polarity had significant effects on total phenolic contents, extracted components, and antioxidant activities. High correlations between phenolic compositions and antioxidant activities of legume extracts were observed. The information is of interest to the nutraceutical food/ingredient industries since legumes are a rich source of antioxidants.

Female mosquitoes are noted for their ability to use odours to locate a host for a blood meal. Two sensory organs contribute to their sense of smell: the maxillary palps, which measure the level of CO2, and the antennae, which detect other host-released odours. To establish the relative importance and interactions of CO2 and other body emissions in freely flying mosquitoes, we presented female yellow fever mosquitoes Aedes aegypti L. with broad plumes of human skin odour and CO2 at natural concentrations and dilutions thereof in a wind tunnel. 3-D video-recorded flight tracks were reconstructed. Activation, flight velocity, upwind turning and source finding waned quickly as skin odours were diluted, whereas in the presence of CO2 these parameters remained unchanged over more than a 100-fold dilution from exhaled concentrations. Although mosquitoes were behaviourally less sensitive to skin odours than to CO2, their sensitivity to skin odours increased transiently by at least fivefold immediately following a brief encounter with a filament of CO2. This sensitization was reflected in flight velocity, track angle, turning rate upon entering and exiting the broad odour plume and, ultimately, in the source-finding rate. In Ae. aegypti, CO2 thus functions as a ;releaser' for a higher sensitivity and responsiveness to skin odours. The initially low responsiveness of mosquitoes to skin odours, their high sensitivity to CO2, and the sensitization of the olfactory circuitry by CO2 are ecologically relevant, because rapidly fluctuating CO2 levels reliably signal a potential host. Possible mechanisms of the instantaneous sensitization are considered.

The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell growth and differentiation are largely unknown. Here, we show that in Arabidopsis thaliana E2FB, accumulation and stability are positively regulated by auxin. Coexpression of E2FB, but not of E2FA, with its dimerization partner A, stimulated cell proliferation in the absence of auxin in tobacco (Nicotiana tabacum) Bright Yellow-2 cells. E2FB regulated the entry into both S- and M-phases, the latter corresponding to the activation of a plant-specific mitotic regulator, CDKB1;1. Increased E2FB levels led to shortened cell cycle duration, elevated cell numbers, and extremely small cell sizes. In the absence of auxin, cells elongated with concomitant increase in their ploidy level, but both were strongly inhibited by E2FB. We conclude that E2FB is one of the key targets for auxin to determine whether cells proliferate or whether they exit the cell cycle, enlarge, and endoreduplicate their DNA.

Targeted therapies for cancer is a rapidly advancing field, but the identification of tumor-specific ligands has proven difficult. Chlorotoxin (CTX) is a small, 36 amino acid neurotoxin isolated from the venom of the Giant Yellow Israeli scorpion Leiurus Quinquestriatus. Interestingly, the peptide has been found to preferentially bind to a variety of human malignancies, but shows little or no binding to normal human tissues. A synthetic version of this peptide (TM-601) has been manufactured and covalently linked to iodine 131 (131I-TM-601) as a means of targeting radiation to tumor cells. Preclinical studies and Phase I clinical trials have been completed in patients with recurrent glioma, a type of malignant brain tumor. These studies demonstrated that intracavitary dosing of 131I-TM-601 appears safe, minimally toxic, and binds malignant glioma with high affinity and for long durations. A Phase II trial of this agent using higher doses of radioactivity and repeated local administrations is underway. In addition, enrolment has begun in a Phase I trial evaluating whether systemically delivered 131I-TM-601 can be used to image metastatic solid tumors and primary gliomas. Due to its small size, selective tumor binding properties, minimal toxicity and relative ease of manipulation, CTX represents a potentially important targeting agent for many cancers.

All mammalian retinae contain rod photoreceptors for low-light vision and cone photoreceptors for daylight and color vision. Most nonprimate mammals have dichromatic color vision based on two cone types with spectrally different visual pigments: a short-wavelength-sensitive (S-)cone and a long-wavelength-sensitive (L-)cone. Superimposed on this basic similarity, there are remarkable differences between species. This article reviews some striking examples. The density ratio of cones to rods ranges from 1:200 in the most nocturnal to 20:1 in a few diurnal species. In some species, the proportion of the spectral cone types and their distribution across the retina deviate from the pattern found in most mammals, including a complete absence of S-cones. Depending on species, the spectral sensitivity of the L-cone pigment may peak in the green, yellow, or orange, and that of the S-cone pigment in the blue, violet, or near-ultraviolet. While exclusive expression of one pigment per cone is the rule, some species feature coexpression of the L- and S-pigment in a significant proportion of their cones. It is widely assumed that all these variations represent adaptations to specific visual needs associated with particular habitats and lifestyles. However, in many cases we have not yet identified the adaptive value of a given photoreceptor arrangement. Comparative anatomy is a fruitful approach to explore the range of possible arrangements within the blueprint of the mammalian retina and to identify species with particularly interesting or puzzling patterns that deserve further scrutiny with physiological and behavioral assays.

Benzidine and related compounds are well known substrates for horseradish peroxidase/H2O2 oxidation. Typically, two different colored products are formed. In this paper, we study the oxidation of 3,5,3',5'-tetramethylbenzidine. The first colored product is a blue charge-transfer complex of the parent diamine and the diimine oxidation product. This species exists in rapid equilibrium with the radical cation. The radical was observed by ESR spectroscopy, and hyperfine splitting constants were determined. Addition of equimolar hydrogen peroxide yields the yellow diimine, which is stable at acid pH. At less than equimolar peroxide, all four species (diamine, radical cation, charge-transfer complex, and diimine) exist in equilibrium. A theoretical analysis of this redox system is presented, including a determination of the extinction coefficients and equilibrium constant for the nonradical species.

We studied physiological properties of glial cells from acute slices of biopsies from patients operated for intractable mesio-temporal lobe epilepsy using whole-cell patch-clamp recordings. Cells were filled with Lucifer Yellow (LY) during recordings to allow morphological reconstruction and immunohistochemical cell identification. Seizure-associated astrocytes had complex, arborized, highly branched processes giving them a stellate appearance, and cells stained intensely for the intermediate filament GFAP as previously reported for 'reactive' astrocytes. GFAP-positive astrocytes from epilepsy biopsies consistently expressed voltage-activated, TTX-sensitive Na+ channels that showed fast activation and inactivation kinetics. Unlike comparison astrocytes, derived from tissues that were not associated with seizure foci, these astrocytes expressed Na+ channels at densities sufficient to generate slow action potentials (spikes) in current clamp studies. In these cells, the ratio of Na+ to K+ conductance was consistently 3-4-fold higher than in comparison human or control rat astrocytes. Four of 17 astrocytes from epilepsy patients versus 14/14 from control rat hippocampus and four of five in comparison human tissue showed a lack of inwardly rectifying K+ currents, which in normal astrocytes are implicated in the control of extracellular K+ levels. These results suggest that astrocytes surrounding seizure foci differ in morphological and physiological properties, and that glial K+ buffering could be impaired at the seizure focus, thus contributing to the pathophysiology of seizures.

The complement system plays a pivotal protective role in the innate immune response to many pathogens including flaviviruses. Flavivirus nonstructural protein 1 (NS1) is a secreted nonstructural glycoprotein that accumulates in plasma to high levels and is displayed on the surface of infected cells but absent from viral particles. Previous work has defined an immune evasion role of flavivirus NS1 in limiting complement activation by forming a complex with C1s and C4 to promote cleavage of C4 to C4b. In this study, we demonstrate a second mechanism, also involving C4 and its active fragment C4b, by which NS1 antagonizes complement activation. Dengue, West Nile, or yellow fever virus NS1 directly associated with C4b binding protein (C4BP), a complement regulatory plasma protein that attenuates the classical and lectin pathways. Soluble NS1 recruited C4BP to inactivate C4b in solution and on the plasma membrane. Mapping studies revealed that the interaction sites of NS1 on C4BP partially overlap with the C4b binding sites. Together, these studies further define the immune evasion potential of NS1 in reducing the functional capacity of C4 in complement activation and control of flavivirus infection.

Adipocyte cell size and number of three adipose depots, gonadal, subcutaneous, and retroperitoneal, were determined in several strains (aA(y), aA(iy), dbdb, obob, and NZO) of adult genetically obese mice, male and female, and in male gold thioglucose-treated mice. Epididymal pad cellularity was determined during development in yellow and viable yellow obese mice and their lean littermates, as well as in the NCS/R mouse. Cell number in the mouse epididymal pad in both lean and genetically obese animals is determined early in development, i.e., before weaning. Cell enlargement is the consistent and usually dominant morphological explanation for adipose depot enlargement in genetic and in gold thioglucose-induced mouse obesity. In some instances, hyperplasia accompanied the hypertrophy, occurring most often in the subcutaneous depot. Cell number in the subcutaneous pad of the obese-hyperglycemic female is four times that of the lean control and represents the most extreme case of hyperplasia observed. In fact, hyperplasia was consistently seen in the obob mouse. A classification for genetic obesity based primarily upon the cellularity characteristics of the adipose depots is proposed.

Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.