Retinal horizontal cells of four rodent species, rat, mouse, gerbil, and guinea pig were examined to determine whether they conform to the basic pattern of two horizontal cell types found in other mammalian orders. Intracellular injections of Lucifer-Yellow were made to reveal the morphologies of individual cells. Immunocytochemistry with antisera against the calcium-binding proteins calbindin D-28k and parvalbumin was used to assess population densities and mosaics. Lucifer-Yellow injections showed axonless A-type and axon-bearing B-type horizontal cells in guinea pig, but revealed only B-type cells in rat and gerbil retinae. Calbindin immunocytochemistry labeled the A- and B-type populations in guinea pig, but only a homogeneous regular mosaic of cells with B-type features in rat, mouse, and gerbil. All calbindin-immunoreactive horizontal cells in the latter species were also parvalbumin-immunoreactive; comparison with Nissl-stained retinae showed that both antisera label all of the horizontal cells. Taken together, the data from cell injections and the population studies provide strong evidence that rat, mouse, and gerbil retinae have only one type of horizontal cell, the axon-bearing B-type, whereas the guinea pig has both A- and B-type cells. Thus, at least three members of the family Muridae differ from other rodents and deviate from the proposed mammalian scheme of horizontal cell types. The absence of A-type cells is apparently not linked to any peculiarities in the photoreceptor populations, and there is no consistent match between the topographic distributions of the horizontal cells and those of the cone photoreceptors or ganglion cells across the four rodent species. However, the cone to horizontal cell ratio is rather similar in the species with and without A-type cells.

Calendar date of the beginning of the growing season at high altitude in the Colorado Rocky Mountains is variable but has not changed significantly over the past 25 years. This result differs from growing evidence from low altitudes that climate change is resulting in a longer growing season, earlier migrations, and earlier reproduction in a variety of taxa. At our study site, the beginning of the growing season is controlled by melting of the previous winter's snowpack. Despite a trend for warmer spring temperatures the average date of snowmelt has not changed, perhaps because of the trend for increased winter precipitation. This disjunction between phenology at low and high altitudes may create problems for species, such as many birds, that migrate over altitudinal gradients. We present data indicating that this already may be true for American robins, which are arriving 14 days earlier than they did in 1981; the interval between arrival date and the first date of bare ground has grown by 18 days. We also report evidence for an effect of climate change on hibernation behavior; yellow-bellied marmots are emerging 38 days earlier than 23 years ago, apparently in response to warmer spring air temperatures. Migrants and hibernators may experience problems as a consequence of these changes in phenology, which may be exacerbated if climate models are correct in their predictions of increased winter snowfall in our study area. The trends we report for earlier formation of permanent snowpack and for a longer period of snow cover also have implications for hibernating species.

Flaviviruses, the human pathogens responsible for dengue fever, West Nile fever, tick-borne encephalitis, and yellow fever, are endemic in tropical and temperate parts of the world. The flavivirus nonstructural protein 1 (NS1) functions in genome replication as an intracellular dimer and in immune system evasion as a secreted hexamer. We report crystal structures for full-length, glycosylated NS1 from West Nile and dengue viruses. The NS1 hexamer in crystal structures is similar to a solution hexamer visualized by single-particle electron microscopy. Recombinant NS1 binds to lipid bilayers and remodels large liposomes into lipoprotein nanoparticles. The NS1 structures reveal distinct domains for membrane association of the dimer and interactions with the immune system and are a basis for elucidating the molecular mechanism of NS1 function.

High expression of epidermal growth factor receptor (EGFR) is found in a variety of solid tumors, including colorectal cancer. EGFR has been identified as a rational target for anticancer therapy. Curcumin, the yellow pigment of turmeric in curry, has received attention as a promising dietary supplement for cancer prevention and treatment. We recently reported that curcumin inhibited the growth of human colon cancer-derived Moser cells by suppressing gene expression of cyclinD1 and EGFR. The aim of the present study was to explore the molecular mechanisms underlying curcumin inhibition of gene expression of EGFR in colon cancer cells. The generality of the inhibitory effect of curcumin on gene expression of EGFR was verified in other human colon cancer-derived cell lines, including Caco-2 and HT-29 cells. Promoter deletion assays and site-directed mutageneses identified a binding site for the transcription factor early growth response-1 (Egr-1) in egfr promoter as a putative curcumin response element in regulating the promoter activity of the gene in Moser cells. Electrophoretic mobility shift assays demonstrated that curcumin significantly reduced the DNA-binding activity of the transcription factor Egr-1 to the curcumin response element. In addition, curcumin reduced the trans-activation activity of Egr-1 by suppressing egr-1 gene expression, which required interruption of the ERK signal pathway and reduction of the level of phosphorylation of Elk-1 and its activity. Taken together, our results demonstrated that curcumin inhibited human colon cancer cell growth by suppressing gene expression of EGFR through reducing the trans-activation activity of Egr-1. These results provided novel insights into the mechanisms of curcumin inhibition of colon cancer cell growth and potential therapeutic strategies for treatment of colon cancer.

It has been contended that limited data exist on sex-difference in immune response with vaccines in humans. However, a comprehensive search of the literature retrieved 97 studies with 14 vaccines influenza (7 studies), hepatitis A (15 studies), hepatitis B (50 studies), pnuemococcal polysaccaride (4 studies), diphtheria (4 studies), rubella (3 studies), measles (2 studies), yellow fever (3 studies), meningococcal A (1 study), meningococcal C (1 study), tetanus (1 study), brucella (1 study), Venezuelan equine encephalitis (1 study) and rabies (4 studies), with sex-difference in humoral (antibody) response. These differences are associated with sex-difference in the clinical efficacy of influenza, hepatitis A, hepatitis B, pneumococcal polysaccharide and diphtheria vaccines and significant adverse reactions with rubella, measles and yellow fever vaccines. The genesis of these differences is uncertain but not entirely related to gonadal hormones (differences are seen in pre-pubertal and post-menopausal subjects not on hormone replacement therapy) or female sex (males had greater serological response for pneumococcal, diphtheria, yellow fever, Venezuelan equine encephalitis and in some studies with rabies vaccine. As sex-difference in humoral immune response was seen with most vaccines which cover the spectrum of mechanisms by which infectious agents cause disease (mucosal replication, viral viraemia, bacterial bacteraemia, toxin production and neuronal invasion), it is mandatory that vaccine trialists recruit a representative sample of females and males to be able to assess sex-differences which may have clinical implications.

Identifying the role of the melanocortin system in regulating energy homeostasis has relied on both genetic and pharmacological studies. The key findings included 1) that the coat color phenotype in the lethal yellow (A(Y)/a) mouse is due to antagonism of the melanocortin-1 receptor (MC1R) by the agouti gene product; 2) the MC3R and MC4R are expressed in CNS centers involved in energy homeostasis, and 3) the combined results of pharmacological studies showing that agouti is an antagonist of the MC4R and transgenic studies showing that inhibition or loss of the MC4R recapitulate the lethal yellow phenotype. Pro-opiomelanocortin (POMC), MC3R, and MC4R knockouts are obese and are now being used to further analyze melanocortin receptor function. The obesity phenotype observed in the MC3R and MC4R knockouts (KO) differ markedly. MC4RKO mice are hyperphagic, do not regulate pathways that increase energy expenditure (diet-induced thermogenesis) and physical activity in response to hyperphagia, and can develop type 2 diabetes. In contrast, MC3R deficient mice are not hyperphagic, have a normal metabolic response to increased energy consumption, and do not develop diabetes. The mechanism underlying the increased adiposity in the MC3R knockout remains unclear, but might be related to changes in nutrient partitioning or physical activity.

Hox genes have been implicated in the evolution of many animal body patterns, but the molecular events underlying trait modification have not been elucidated. Pigmentation of the posterior male abdomen is a recently acquired trait in the Drosophila melanogaster lineage. Here, we show that the Abdominal-B (ABD-B) Hox protein directly activates expression of the yellow pigmentation gene in posterior segments. ABD-B regulation of pigmentation evolved through the gain of ABD-B binding sites in a specific cis-regulatory element of the yellow gene of a common ancestor of sexually dimorphic species. Within the melanogaster species group, male-specific pigmentation has subsequently been lost by at least three different mechanisms, including the mutational inactivation of a key ABD-B binding site in one lineage. These results demonstrate how Hox regulation of traits and target genes is gained and lost at the species level and have general implications for the evolution of body form at higher taxonomic levels.

Curcumin, the yellow color pigment of turmeric, is produced industrially from turmeric oleoresin. The mother liquor after isolation of curcumin from oleoresin contains approximately 40% oil. The oil was extracted from the mother liquor using hexane at 60 degrees C, and the hexane extract was separated into three fractions using silica gel column chromatography. These fractions were tested for antibacterial activity by pour plate method against Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Fraction II eluted with 5% ethyl acetate in hexane was found to be most active fraction. The turmeric oil, fraction I, and fraction II were analyzed by GC and GC-MS. ar-Turmerone, turmerone, and curlone were found to be the major compounds present in these fractions along with other oxygenated compounds.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are processed by the ribonuclease Dicer from distinct precursors, double-stranded RNA (dsRNA) and hairpin RNAs, respectively, although either may guide RNA silencing via a similar complex. The siRNA pathway is antiviral, whereas an emerging role for miRNAs is in the control of development. Here, we describe a virulence factor encoded by turnip yellow mosaic virus, p69, which suppresses the siRNA pathway but promotes the miRNA pathway in Arabidopsis thaliana. p69 suppression of the siRNA pathway is upstream of dsRNA and is as effective as genetic mutations in A. thaliana genes involved in dsRNA production. Possibly as a consequence of p69 suppression, p69-expressing plants contained elevated levels of a Dicer mRNA and of miRNAs as well as a correspondingly enhanced miRNA-guided cleavage of two host mRNAs. Because p69-expressing plants exhibited disease-like symptoms in the absence of viral infection, our findings suggest a novel mechanism for viral virulence by promoting the miRNA-guided inhibition of host gene expression.

Knowledge of the thickness of sections is important for proper interpretation of electron micrographs. Therefore, the thicknesses of sections of n-butyl methacrylate polymer were determined by ellipsometry, and correlated with the color shown in reflected light. The results are: gray, thinner than 60 mmicro; silver, 60 to 90 mmicro; gold, 90 to 150 mmicro; purple, 150 to 190 mmicro; blue, 190 to 240 mmicro; green, 240 to 280 mmicro; and yellow, 280 to 320 mmicro. These results agree well with optical theory and with previous published data for thin films. Sections, after cutting, are 30 to 40 per cent shorter than the face of the block from which they were cut. Only a small improvement results from allowing the sections to remain in the collecting trough at room temperature. Heating above room temperature, however, reduces this shortening, with a corresponding improvement in dimensions and spatial relationships in the sections. When the thickness of the section is considered in interpreting electron micrographs instead of considering the section to be two-dimensional, a more accurate interpretation is possible. The consideration of electron micrographs as arising from projections of many profiles from throughout the whole thickness of the section explains the apparent lack of continuity often observed in serial sections. It is believed that serial sections are actually continuous, but that the change in size of structure through the thickness of one section and the consideration of only the largest profile shown in the micrograph can account for the lack of continuity previously observed.

The 5'-terminal noncoding region sequences were determined for the genome RNAs of seven strains of St. Louis encephalitis virus (SLEV) and one strain of West Nile virus (WNV) using a single synthetic cDNA primer complementary to the 5'-terminus of the coding region of a strain of WNV RNA. The 5'-terminal sequences obtained for the SLEV and WNV RNAs were compared with published sequences for yellow fever virus (YFV), Murray Valley encephalitis virus (MVEV), and dengue virus. While only short regions within the 5'-noncoding sequence were conserved among different flavivirus RNAs, significant homology was observed in this region among members of the same flavivirus subgroup and almost complete conservation was observed between different strains of the same virus. For example, seven strains of SLE, isolated from different geographic locations over a 17-year period and differing in their neurovirulence phenotype, contained only two to four nucleotide changes in the 5'-noncoding region. Interestingly, each of three low-virulence strains shared the same unique base substitution at position 16. Secondary structures predicted to be formed by the 5'-termini of each of the different flavivirus genome RNAs were of similar size and shape, in each case consisting of a stem with a small top loop and a larger side loop. The prediction of a common structure among a number of different flaviviruses, despite the lack of extensive sequence homology, suggests that this secondary structure is functionally important. An additional stem and loop structure is predicted to be formed in the region spanning the translation initiation codon. This structure showed significantly less conservation of size and shape than the 5'-terminal secondary structure.

X-ray and neutron crystallographic techniques provide complementary information on the structure and function of biological macromolecules. X-ray and neutron (XN) crystallographic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. The XN approach for complete (including hydrogen) macromolecular structure analysis provides more accurate and complete structures, as demonstrated for diisopropyl fluorophosphatase, photoactive yellow protein and human aldose reductase. Furthermore, this method has several practical advantages, including the easier determination of the orientation of water molecules, hydroxyl groups and some amino-acid side chains.

Models of the differentiation of memory CD8+ T cells that replicate during secondary infections differ over whether such cells had acquired effector function during primary infections. We created a transgenic mouse line that permits mapping of the fate of granzyme B (gzmB)-expressing CD8+ T cells and their progeny by indelibly marking them with enhanced yellow fluorescent protein (EYFP). Virus-specific CD8+ T cells express gzmB within the first 2 days of a primary response to infection with influenza, without impairment of continued primary clonal expansion. On secondary infection, virus-specific CD8+ T cells that became EYFP+ during a primary infection clonally expand as well as all virus-specific CD8+ T cells. Thus, CD8+ T cells that have acquired an effector phenotype during primary infection may function as memory cells with replicative function.

We have determined the complete sequence of the RNA of dengue 2 virus (S1 candidate vaccine strain derived from the PR-159 isolate) with the exception of about 15 nucleotides at the 5' end. The genome organization is the same as that deduced earlier for other flaviviruses and the amino acid sequences of the encoded dengue 2 proteins show striking homology to those of other flaviviruses. The overall amino acid sequence similarity between dengue 2 and yellow fever virus is 44.7%, whereas that between dengue 2 and West Nile virus is 50.7%. These viruses represent three different serological subgroups of mosquito-borne flaviviruses. Comparison of the amino acid sequences shows that amino acid sequence homology is not uniformly distributed among the proteins; highest homology is found in some domains of nonstructural protein NS5 and lowest homology in the hydrophobic polypeptides ns2a and 2b. In general the structural proteins are less well conserved than the nonstructural proteins. Hydrophobicity profiles, however, are remarkably similar throughout the translated region. Comparison of the dengue 2 PR-159 sequence to partial sequence data from dengue 4 and another strain of dengue 2 virus reveals amino acid sequence homologies of about 64 and 96%, respectively, in the structural protein region. Thus as a general rule for flaviviruses examined to date, members of different serological subgroups demonstrate 50% or less amino acid sequence homology, members of the same subgroup average 65-75% homology, and strains of the same virus demonstrate greater than 95% amino acid sequence similarity.

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.

Hepatic fibrogenesis occurs as a wound-healing process after many forms of chronic liver injury. Hepatic fibrosis ultimately leads to cirrhosis if not treated effectively. During liver injury, quiescent hepatic stellate cells (HSC), the most relevant cell type, become active and proliferative. Oxidative stress is a major and critical factor for HSC activation. Activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) inhibits the proliferation of nonadipocytes. The level of PPAR-gamma is dramatically diminished along with activation of HSC. Curcumin, the yellow pigment in curry, is a potent antioxidant. The aims of this study were to evaluate the effect of curcumin on HSC proliferation and to begin elucidating underlying mechanisms. It was hypothesized that curcumin might inhibit the proliferation of activated HSC by inducing PPAR-gamma gene expression and reviving PPAR-gamma activation. Our results indicated that curcumin significantly inhibited the proliferation of activated HSC and induced apoptosis in vitro. We demonstrated, for the first time, that curcumin dramatically induced the gene expression of PPAR-gamma and activated PPAR-gamma in activated HSC. Blocking its trans-activating activity by a PPAR-gamma antagonist markedly abrogated the effects of curcumin on inhibition of cell proliferation. Our results provide a novel insight into mechanisms underlying the inhibition of activated HSC growth by curcumin. The characteristics of curcumin, including antioxidant potential, reduction of activated HSC growth, and no adverse health effects, make it a potential antifibrotic candidate for prevention and treatment of hepatic fibrosis.

In yeast, chronological senescence (CS) is defined as loss of viability in stationary culture. Although its relevance to the organismal aging remained unclear, yeast CS was one of the most fruitful models in aging research. Here we described a mammalian replica of yeast CS: loss of viability of overgrown "yellow" cancer cell culture. In a density and time (chronological)-dependent manner, cell culture loses the ability to re-grow in fresh medium. Rapamycin dramatically decelerated CS. Loss of viability was caused by acidification of the medium by lactic acid (lactate). Rapamycin decreased production of lactate, making conditioned medium (CM) less deadly. Both deadly CM and lactate caused loss of viability in low cell density, not preventable by either rapamycin or additional glucose. Also, NAC, LY294002, U0126, GSK733, which all indirectly inhibit mTOR and have been shown to suppress the senescent phenotype in traditional models of mammalian cell senescence, also decreased lactate production and decelerated CS. We discuss that although CS does not mimic organismal aging, the same signal transduction pathways that drive CS also drive aging.

The motile, alkalophilic, and extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila is positively photophobotactic. This response results in the accumulation of bacteria in light spots (E. Hustede, M. Liebergesell, and H. G. Schlegel, Photochem. Photobiol. 50:809-815, 1989; D. E. McRee, J. A. Tainer, T. E. Meyer, J. Van Beeumen, M. A. Cusanovich, and E. D. Getzoff, Proc. Natl. Acad. Sci. USA 86:6533-6537, 1989; also, this work). In this study, we demonstrated that E. halophila is also negatively phototactic. Video analysis of free-swimming bacteria and the formation of cell distribution patterns as a result of light-color boundaries in an anaerobic suspension of cells revealed the existence of a repellent response toward intense (but nondamaging) blue light. In the presence of saturating background photosynthetic light, an increase in the intensity of blue light induced directional switches, whereas a decrease in intense blue light gave rise to suppression of these reversals. To our knowledge, this is the first report of a true repellent response to light in a free-swimming eubacterium, since the blue light response in Escherichia coli and Salmonella typhimurium (B. L. Taylor and D. E. Koshland, Jr., J. Bacteriol. 123:557-569, 1975), which requires an extremely high light intensity, is unlikely to be a sensory process. The wavelength dependence of this negative photoresponse was determined with narrow band pass interference filters. It showed similarity to the absorption spectrum of the photoactive yellow protein from E. halophila.

OBJECTIVE

Intercellular Ca(2+) wave propagation is a distinct form of cell-cell communication. In corneal endothelial cells, intercellular Ca(2+) wave propagation evoked by a point mechanical stimulus (PMS) is partially mediated by adenosine triphosphate (ATP) release and subsequent activation of P2Y receptors. This study was conducted to investigate the possibility that extrajunctional connexons (hemichannels) play a role in ATP release during PMS-induced Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs).

METHODS

A Ca(2+) wave was evoked by a PMS applied to a single cell in a monolayer of cultured BCECs. Changes in [Ca(2+)](i) in the mechanically stimulated cell (MS cell) and in the neighboring (NB) cells were visualized by fluorescence imaging using the Ca(2+)-sensitive dye Fluo-4. From these images, the maximum normalized fluorescence (NF), the percentage of responsive cells (%RC), and the total area of cells reached by the Ca(2+) wave (active area [AA], in square micrometers) were calculated. Intercellular dye transfer, generally attributed to gap junctional coupling, was assessed by fluorescence recovery after photobleaching (FRAP) using 6-carboxyfluorescein diacetate. Opening of hemichannels was investigated by measuring cellular uptake of the fluorescent dye Lucifer yellow, which is known to permeate hemichannels. ATP release was measured by luciferin-luciferase bioluminescence.

RESULTS

Flufenamic acid (FFA; 50 microM) and the connexin mimetic peptide Gap26 (300 microM), known blockers of hemichannels, significantly reduced AA in confluent monolayers as well as in contact-free cells. Neither FFA nor Gap26 affected the FRAP, indicating that reduction in AA of the PMS-induced wave by these agents is not due to a block of gap junction channels. FFA as well as Gap26 inhibited the increase in AA of the wave that was observed when cells were pretreated with the ectonucleotidase inhibitor ARL-67156 (100 microM). These findings suggest that the hemichannel blockers reduce the Ca(2+) wave propagation by inhibiting ATP release. Consistent with this finding, PMS or exposure to Ca(2+)-free solution (a maneuver known to induce the opening of hemichannels) led to ATP release; moreover, the release was inhibited by the hemichannel blockers. The extracellular ATP levels in response to both PMS and extracellular Ca(2+) removal were strongly enhanced by ARL-67156, and this effect was inhibited by FFA as well as by Gap26. Moreover, pretreatment of subconfluent BCEC monolayers with FFA or Gap26 inhibited the uptake of Lucifer yellow induced by removal of extracellular Ca(2+).

CONCLUSIONS

Hemichannels contribute to ATP release on mechanical stimulation in BCECs. The released ATP contributes to propagation of the Ca(2+) wave.

Although neocortical GABAergic (gamma-aminobutyric acidergic) interneurons have been the focus of intense study, especially in the rat, a consensus view of the functional diversity and organization of inhibitory cortical neurons has not yet been achieved. To better analyze GABAergic neurons in the rat, we used a bacterial artificial chromosome (BAC) construct and established 2 lines of transgenic rats that coexpress Venus, a yellow fluorescent protein, with the vesicular GABA transporter. The brain GABA content from both transgenic lines was similar to the level found in wild-type rats. In the frontal cortex, Venus was expressed in >95% of GABAergic neurons, most of which also expressed at least one of 6 biochemical markers, including alpha-actitin-2, which preferentially labeled late-spiking neurogliaform cells. Taking advantage of the fact that Venus expression allows for targeted recording from all classes of nonpyramidal cells, irrespective of their somatic morphologies, we demonstrated that fast-spiking neurons, which were heterogeneous in somatic size as well as vertical dendritic projection, had relatively uniform horizontal dimensions, suggesting a cell type-specific columnar input territory. Our data demonstrate the benefits of VGAT-Venus rats for investigating GABAergic circuits, as well as the feasibility of using BAC technology in rats to label subsets of specific, genetically defined neurons.

We performed intracellular recording with Lucifer yellow dye microinjection to investigate the cellular pathway(s) by which constriction and dilation are conducted along the wall of arterioles (diameter 47 +/- 1 microns, n = 63) supplying blood flow to the cheek pouch of anesthetized hamsters. At rest, membrane potential (Em) of endothelial (-36 +/- 1 mV) and smooth muscle (-35 +/- 1 mV) cells was not different. Micropipette delivery of norepinephrine (NE) or phenylephrine (PE) produced smooth muscle cell depolarization (5-41 mV) and vasoconstriction (7-49 microns) at the site of release and along the arteriole with no effect on Em of endothelial cells. KCl produced conduction of depolarization and vasoconstriction with similar electrical kinetics in endothelial and smooth muscle cells. Acetylcholine triggered conduction of vasodilation (2-25 microns) and hyperpolarization (3-33 mV) along both cell layers; in smooth muscle, this change in Em was prolonged and followed by a transient depolarization. These cell-specific electrophysiological recordings uniquely illustrate that depolarization and constriction are initiated and conducted along smooth muscle, independent of the endothelium. Furthermore, conduction of vasodilation is explained by the spread of hyperpolarization along homologously coupled endothelial and smooth muscle cells, with distinctive responses between cell layers. The discontinuity between endothelium and smooth muscle indicates that these respective pathways are not electrically coupled during blood flow control.

Gold is known as a shiny, yellow noble metal that does not tarnish, has a face centred cubic structure, is non-magnetic and melts at 1336 K. However, a small sample of the same gold is quite different, providing it is tiny enough: 10 nm particles absorb green light and thus appear red. The melting temperature decreases dramatically as the size goes down. Moreover, gold ceases to be noble, and 2-3 nm nanoparticles are excellent catalysts which also exhibit considerable magnetism. At this size they are still metallic, but smaller ones turn into insulators. Their equilibrium structure changes to icosahedral symmetry, or they are even hollow or planar, depending on size. The present tutorial review intends to explain the origin of this special behaviour of nanomaterials.

The functional properties of proteins [capsid protein (CP), V1, and C4] potentially involved with movement of the monopartite begomovirus, Tomato yellow leaf curl virus (TYLCV), were investigated using microinjection of Escherichia coli expressed proteins and transient expression of GFP fusion proteins. The TYLCV CP localized to the nucleus and nucleolus and acted as a nuclear shuttle, facilitating import and export of DNA. Thus, the CP serves as the functional homolog of the bipartite begomovirus BV1. The TYLCV V1 localized around the nucleus and at the cell periphery and colocalized with the endoplasmic reticulum, whereas C4 was localized to the cell periphery. Together, these patterns of localization were similar to that of the bipartite begomovirus BC1, known to mediate cell-to-cell movement. However, in contrast to BC1, V1 and C4, alone or in combination, had a limited capacity to move and mediate macromolecular trafficking through mesophyll or epidermal plasmodesmata. Immunolocalization and in situ PCR experiments, conducted with tomato plants at three stages of development, established that TYLCV infection was limited to phloem cells of shoot apical, leaf, stem, and floral tissues. Thus, the V1 and/or C4 may be analogs of the bipartite begomovirus BC1 that have evolved to mediate TYLCV movement within phloem tissue.

Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10(-6)-10(-3)-s time range, by using fluorescence correlation spectroscopy at 10(-8) M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over approximately 6 x 10(2) to 5 x 10(6) W/cm(2). These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of approximately 10(-3). Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-microseconds time range. The independence of these two bright-dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.