Fast spiking cells in the CA1 region of the rat hippocampus were revealed as gamma-aminobutyric acid (GABA)ergic non-pyramidal cells containing the calcium-binding protein parvalbumin by intracellular injection of Lucifer yellow in vitro in combination with postembedding parvalbumin immunohistochemistry.

The objective of this study was to assess the quality of the current intakes of fruits and vegetables compared to the Dietary Guidelines for Americans in US children and adolescents and identify factors related to low fruit and vegetable intake. This descriptive study examined differences in fruit and vegetable intakes by age, sex, ethnicity, poverty level, body mass index, and food security status utilizing data from the 1999-2002 National Health and Nutrition Examination Survey. Six thousand five hundred thirteen children and adolescents ages 2 to 18 years, who were respondents to the 1999-2002 National Health and Nutrition Examination Survey. Mean fruit and vegetable intakes were computed using 24-hour recalls for individuals and compared using analysis of variance. Leading contributors to fruit and vegetable intake were identified using frequency analysis. Children aged 2 to 5 years had significantly higher total fruit and juice intakes than 6- to 11- and 12- to 18-year-olds. Total vegetable and french fry intake was significantly higher among 12- to 18-year-old adolescents. Regarding sex differences, boys consumed significantly more fruit juice and french fries than girls. In addition, non-Hispanic African-American children and adolescents consumed significantly more dark-green vegetables and fewer mean deep-yellow vegetables than Mexican-American and non-Hispanic white children and adolescents. Total fruit consumption also differed significantly among race/ethnicities and household income. Children and adolescents most at risk for higher intakes of energy-dense fruits and vegetables (fruit juice and french fries) were generally boys, and adolescents, at risk for overweight or overweight and living in households below 350% of the poverty level.

Positive strand RNA viral genomes are unique in the viral world in serving a dual role as mRNA and replicon. Since the origin of the minus-strand RNA replication intermediate is at the 3'-end of the genome, the 3'-untranslated region (UTR) clearly plays a role in viral RNA replication. The messenger role of this same RNA likely places functional demands on the 3'-UTR to serve roles typical of cellular mRNAs, including the regulation of RNA stability and translation. Current understanding indicates varied roles for positive strand RNA viral 3'-UTRs, with the dominant roles differing between viruses. Three case studies are discussed: turnip yellow mosaic virus RNA, whose 3' tRNA mimicry is thought to negatively regulate minus strand synthesis; brome mosaic virus, whose 3'-UTR contains a unique promoter element directing minus strand synthesis; and tobacco mosaic virus, whose 3'-UTR contains an enhancer of translational expression.

Caspase-3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase-3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase-3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e. CFP-DEVD-YFP). We demonstrate that, once initiated, the activation of caspase-3 is a very rapid process, taking 5 min or less to reach completion. Furthermore, this process occurs almost simultaneously with a depolarization of the mitochondrial membrane potential. These events occur just prior to the characteristic morphological changes associated with apoptosis. Our results clearly demonstrate that, once initiated, the commitment of cells to apoptosis is a remarkably rapid event when visualized at the single cell level.

In vivo 1H NMR spectra of small volumes-of-interest (VOI) were localized in human soleus muscle (8 ml) and compared with volume selective spectra of subcutaneous fat tissue and femoral yellow bone marrow (2 ml). All examinations were performed by the double spin echo (PRESS) localization technique. To provide comparability, spectra of different tissues were recorded using identical sequence timing. Clearly improved resolution of the lipid signals of muscle tissue was obtained using long echo times TE>> 200 ms. The spectra of muscle tissue exhibit lipid signals that stem from two compartments with a difference of their resonance frequencies of about 0.2 ppm (Larmor frequency difference 12-13 Hz at 1.5 T). The existence of two fatty acid compartments is supported by measurements of the relaxation times and line shape analysis. Both compartments contain fatty acids or triglycerides with similar composition. Probably one compartment corresponds to fat cells within muscle tissue, the other compartment with lower Larmor frequency is located within muscle cells.

The cytochrome oxidase-rich patches or blobs of the monkey striate cortex have been shown to contain cells that have unoriented receptive fields, many of which are color selective. We studied the functional organization of color opponency in the blob regions of the parafoveal representation of the visual cortex. We also examined the patterns of connectivity among blob and nonblob cells by multiple electrode penetrations and cross-correlation analysis. Some of the color-selective cells in the blobs exhibited receptive fields that were similar to those found in the parvocellular layers of the lateral geniculate nucleus (LGN): one type exhibited center-surround spatial and chromatic opponency corresponding to the Type I cell found in the LGN; another had center-only chromatic opponency, corresponding to the Type II cell of the LGN. A blob color-selective cell with no LGN counterpart had center color opponency with a nonchromatically opponent surround antagonism. We termed this cell the "modified Type II" cell. Contrary to previous reports, few true double color-opponent cells were found. Some blob cells previously characterized as double opponent probably belong to our modified Type II category and, unlike true double opponent cells, do not respond well to isoluminant color boundaries. Occasional color-selective oriented cells were either intermixed or in close proximity to blob cells. Neighboring electrode penetrations within the same blob yielded cells of the same color opponency, either red versus green or blue versus yellow, suggesting that individual blobs are dedicated to processing one color opponency. Blobs dedicated to red/green color opponency were 3 times more numerous than blue/yellow blobs. Furthermore, the cells in layer 4C lying beneath blobs of a given color opponency had identical color opponency to the overlying cells in blobs. Cross-correlation analysis of pairs of nonblob, oriented cells in the superficial layers showed interactions between cells with matched orientation and eye preference, at varying horizontal separations. Such interactions are consistent with anatomically demonstrated clustered horizontal connections. Positive cross-correlograms were found between blob cells in the same and in adjacent blobs when the cells' receptive field type, color opponency, and ocular dominance matched. Correlograms also indicated monosynaptic connections from Type II to modified Type II cells of the same color opponency, suggesting that Type II cells may contribute to the construction of the modified Type II fields in the cortex.(ABSTRACT TRUNCATED AT 400 WORDS)

With the emergence of Staphylococcus aureus as a prominent pathogen in community and healthcare settings, there is a growing need for effective reporter tools to facilitate physiology and pathogenesis studies. Fluorescent proteins are ideal as reporters for their convenience in monitoring gene expression, performing host interaction studies, and monitoring biofilm growth. We have developed a suite of fluorescent reporter plasmids for labeling S. aureus cells. These plasmids encode either green fluorescent protein (GFP) or higher wavelength reporter variants for yellow (YFP) and red (mCherry) labeling. The reporters were placed under control of characterized promoters to enable constitutive or inducible expression. Additionally, plasmids were assembled with fluorescent reporters under control of the agr quorum-sensing and sigma factor B promoters, and the fluorescent response with wildtype and relevant mutant strains was characterized. Interestingly, reporter expression displayed a strong dependence on ribosome binding site (RBS) sequence, with the superoxide dismutase RBS displaying the strongest expression kinetics of the sequences examined. To test the robustness of the reporter plasmids, cell imaging was performed with fluorescence microscopy and cell populations were separated using florescence-activated cell sorting (FACS), demonstrating the possibilities of simultaneous monitoring of multiple S. aureus properties. Finally, a constitutive YFP reporter displayed stable, robust labeling of biofilm growth in a flow-cell apparatus. This toolbox of fluorescent reporter plasmids will facilitate cell labeling for a variety of different experimental applications.

The Mycobacterium avium complex, only rarely described as an invasive pathogen in humans, has recently been reported to frequently cause disseminated disease in patients with the acquired immune deficiency syndrome. Between February 1981 and February 1984 at Memorial Sloan-Kettering Cancer Center, 30 patients with acquired immune deficiency syndrome, 3 patients with leukemia, and 2 patients with congenital severe combined immunodeficiency syndrome developed disseminated M. avium complex infection. Mycobacteria were often found in multiple sites both antemortem and postmortem. Blood cultures were a reliable method for detecting disseminated infection, and the new lysis blood culture systems provided an efficient technique for determining the number of organisms per milliliter of blood. Acid-fast stains and cultures of fecal specimens were also helpful in diagnosing infection. Most of the mycobacteria were serovar 4 (77%), and most (86%) produced a deep yellow pigment. All isolates were susceptible to standard concentrations of clofazimine, cycloserine, and ansamycin, but tended to be resistant to isoniazid, streptomycin, ethambutol, ethionamide, and rifampin.

The mesolimbic dopamine system, which arises in the ventral tegmental area (VTA), is an important neural substrate for opiate reinforcement and addiction. Chronic exposure to opiates is known to produce biochemical adaptations in this brain region. We now show that these adaptations are associated with structural changes in VTA dopamine neurons. Individual VTA neurons in paraformaldehyde-fixed brain sections from control or morphine-treated rats were injected with the fluorescent dye Lucifer yellow. The identity of the injected cells as dopaminergic or nondopaminergic was determined by immunohistochemical labeling of the sections for tyrosine hydroxylase. Chronic morphine treatment resulted in a mean approximately 25% reduction in the area and perimeter of VTA dopamine neurons. This reduction in cell size was prevented by concomitant treatment of rats with naltrexone, an opioid receptor antagonist, as well as by intra-VTA infusion of brain-derived neurotrophic factor. In contrast, chronic morphine treatment did not alter the size of nondopaminergic neurons in the VTA, nor did it affect the total number of dopaminergic neurons in this brain region. The results of these studies provide direct evidence for structural alterations in VTA dopamine neurons as a consequence of chronic opiate exposure, which could contribute to changes in mesolimbic dopamine function associated with addiction.

Spinal astrocytes are coupled by connexin (Cx) gap junctions and express pannexin 1 (Px1) and purinergic receptors. Fibroblast growth factor 1 (FGF-1), which is released in spinal cord injury, activated spinal astrocytes in culture, induced secretion of ATP, and permeabilized them to relatively large fluorescent tracers [ethidium (Etd) and lucifer yellow (LY)] through "hemichannels" (HCs). HCs can be formed by connexins or pannexins; they can open to extracellular space or can form gap junction (GJ) channels, one HC from each cell. (Pannexins may not form gap junctions in mammalian tissues, but they do in invertebrates). HC types were differentiated pharmacologically and by Px1 knockdown with siRNA and by use of astrocytes from Cx43 knockout mice. Permeabilization was reduced by apyrase (APY), an ATPase, and by P2X(7) receptor antagonists, implicating secretion of ATP and autocrine and/or paracrine action. Increased permeability of cells exposed to FGF-1 or ATP for 2 h was mediated largely by Px1 HCs activated by P2X(7) receptors. After a 7-h treatment, the permeability was mediated by both Cx43 and Px1 HCs. FGF-1 also caused reduction in gap junctional communication. Botulinum neurotoxin A, a blocker of vesicular release, reduced permeabilization when given 30 min before FGF-1 application, but not when given 1 h after FGF-1. We infer that ATP is initially released from vesicles and then it mediates continued release by action on P2X(7) receptors and opening of HCs. These changes in HCs and gap junction channels may promote inflammation and deprive neurons of astrocyte-mediated protection in spinal cord trauma and neurodegenerative disease.

Several different rodent models are available for metabolic studies on the development of diabetes. Although the abnormalities associated with each diabetes type have many features in common, the documentation of several different genes being involved makes it unlikely that the various syndromes will be reduced to a single disturbance in one metabolic pathway. The severity of the diabetes produced depends on the interaction of the individual mutation with genetic factors in the inbred background of the host. Establishing the nature of these gene-host interactions in rodents should aid us in understanding similar interactions that occur in human diabetes. The development of the syndrome in most models is similar and includes hyperinsulinemia, hyperphagia, and attempts at increasing insulin supply by beta-cell hyperplasia and hypertrophy in the early stages. Hyperglycemia, obesity, and severe diabetes are secondary features that result from a combination of insulin resistance and a failure to sustain the secretion of the large amounts of insulin. Most models utilize ingested food and stored food reserves more efficiently. This increased metabolic efficiency extends to heterozygotes that are normal in all respects having only one dose of the deleterious gene. Establishing this increased metabolic efficiency in heterozygotes lends credence to the thrifty gene hypothesis of diabetes and suggests a mechanism whereby some deleterious diabetes genes may be favored in the human population. The best studied mouse models, and those for which the most complete information is available, are those caused by single genes, e.g., yellow, obese, diabetes, tubby, and fat. In the other models, the mode of inheritance is either polygenic or otherwise unclear, features which interfere with the interpretation of the data. This report briefly summarizes the developing syndrome in each model, points out any differences, and suggests the most appropriate areas where future research should be most productive in the light of contemporary studies.

The bimolecular fluorescence complementation (BiFC) assay has been widely accepted for studying in vivo detection of protein-protein interactions in several organisms. To facilitate the application of the BiFC assay to yeast research, we have created a series of plasmids that allow single-step, PCR-based C- or N-terminal tagging of yeast proteins with yellow fluorescent protein fragments for BiFC assay. By examination of several interacting proteins (Sis1-Sis1, Net1-Sir2, Cet1-Cet1 and Pho2-Pho4), we demonstrate that the BiFC assay can be used to reliably analyse the occurrence and subcellular localization of protein-protein interactions in living yeast cells. The sequences for the described plasmids were submitted to the GenBank under Accession Nos: EF210802, pFA6a-VN-His3MX6; EF210803, pFA6a-VC-His3MX6; EF210804, pFA6a-VN-TRP1; EF210807, pFA6a-VC-TRP1; EF210808, pFA6a-VN-kanMX6; EF210809, pFA6a-VC-kanMX6; EF210810, pFA6a-His3MX6-P(GAL1)-VN; EF210805, pFA6a-His3MX6-P(GAL1)-VC; EF210806, pFA6a-TRP1-P(GAL1)-VN; EF210811, pFA6a-TRP1-P(GAL1)-VC; EF210812, pFA6a-kanMX6-P(GAL1)-VN; EF210813, pFA6a-kanMX6-P(GAL1)-VC; EF521883, pFA6a-His3MX6-P(CET1)-VN; EF521884, pFA6a-His3MX6-P(CET1)-VC; EF521885, pFA6a-TRP1-P(CET1)-VN; EF521886, pFA6a-TRP1-P(CET1)-VC; EF521887, pFA6a-kanMX6-P(CET1)-VN; EF521888, pFA6a-kanMX6-P(CET1)-VC.

Dengue virus is the most widespread geographically of the arboviruses and a major public health threat in the tropics and subtropics. Scientific advances in recent years have provided new insights about the pathogenesis of more severe disease and novel approaches into the development of antiviral compounds and dengue vaccines. Phylogenetic studies show an association between specific subtypes (within serotypes) and severity of dengue. The lack of association between maternal antibodies and development of severe dengue in infants in a recent study has called for the rethinking or refinement of the current antibody-dependent enhancement theory of dengue hemorrhagic syndrome in infancy. Such studies should stimulate new directions of research into mechanisms responsible for the development of severe dengue. The life cycle of dengue virus readily shows that virus entry and replication can be targeted by small molecules. Advances in a mouse model (AG 129 mice) have made it easier to test such antiviral compounds. The efforts to find specific dengue inhibitors are intensifying and the tools to evaluate the efficacy of new drugs are now in place for rapid translation into trials in humans. Furthermore, several dengue vaccine candidates are in development, of which the chimeric dengue/yellow fever vaccine has now entered phase 3 trials. Until the availability of a licensed vaccine, disease surveillance and vector population control remain the mainstay of dengue prevention.

The epigenetic effects on DNA methylation, histone modification, and expression of non-coding RNAs (including microRNAs) of environmental chemicals such as bisphenol A (BPA) and phthalates have expanded our understanding of the etiology of human complex diseases such as cancers and diabetes. Multiple lines of evidence from in vitro and in vivo models have established that epigenetic modifications caused by in utero exposure to environmental toxicants can induce alterations in gene expression that may persist throughout life. Epigenetics is an important mechanism in the ability of environmental chemicals to influence health and disease, and BPA and phthalates are epigenetically toxic. The epigenetic effect of BPA was clearly demonstrated in viable yellow mice by decreasing CpG methylation upstream of the Agouti gene, and the hypomethylating effect of BPA was prevented by maternal dietary supplementation with a methyl donor like folic acid or the phytoestrogen genistein. Histone H3 was found to be trimethylated at lysine 27 by BPA effect on EZH2 in a human breast cancer cell line and mice. BPA exposure of human placental cell lines has been shown to alter microRNA expression levels, and specifically, miR-146a was strongly induced by BPA treatment. In human breast cancer MCF7 cells, treatment with the phthalate BBP led to demethylation of estrogen receptor (ESR1) promoter-associated CpG islands, indicating that altered ESR1 mRNA expression by BBP is due to aberrant DNA methylation. Maternal exposure to phthalate DEHP was also shown to increase DNA methylation and expression levels of DNA methyltransferases in mouse testis. Further, some epigenetic effects of BPA and phthalates in female rats were found to be transgenerational. Finally, the available new technologies for global analysis of epigenetic alterations will provide insight into the extent and patterns of alterations between human normal and diseased tissues. In vitro models such as human embryonic stem cells may be extremely useful in bettering the understanding of epigenetic effects on human development, health and disease, because the formation of embryoid bodies in vitro is very similar to the early stage of embryogenesis.

The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.

Cellular homeostasis in neurons requires that the synthesis and anterograde axonal transport of protein and membrane be balanced by their degradation and retrograde transport. To address the nature and regulation of retrograde transport in cultured sympathetic neurons, I analyzed the behavior, composition, and ultrastructure of a class of large, phase-dense organelles whose movement has been shown to be influenced by axonal growth (Hollenbeck, P. J., and D. Bray. 1987. J. Cell Biol. 105:2827-2835). In actively elongating axons these organelles underwent both anterograde and retrograde movements, giving rise to inefficient net retrograde transport. This could be shifted to more efficient, higher volume retrograde transport by halting axonal outgrowth, or conversely shifted to less efficient retrograde transport with a larger anterograde component by increasing the intracellular cyclic AMP concentration. When neurons were loaded with Texas red-dextran by trituration, autophagy cleared the label from an even distribution throughout the neuronal cytosol to a punctate, presumably lysosomal, distribution in the cell body within 72 h. During this process, 100% of the phase-dense organelles were fluorescent, showing that they contained material sequestered from the cytosol and indicating that they conveyed this material to the cell body. When 29 examples of this class of organelle were identified by light microscopy and then relocated using correlative electron microscopy, they had a relatively constant ultrastructure consisting of a bilamellar or multilamellar boundary membrane and cytoplasmic contents, characteristic of autophagic vacuoles. When neurons took up Lucifer yellow, FITC-dextran, or Texas red-ovalbumin from the medium via endocytosis at the growth cone, 100% of the phase-dense organelles became fluorescent, demonstrating that they also contain products of endocytosis. Furthermore, pulse-chase experiments with fluorescent endocytic tracers confirmed that these organelles are formed in the most distal region of the axon and undergo net retrograde transport. Quantitative ratiometric imaging with endocytosed 8-hydroxypyrene-1,3,6-trisulfonic acid showed that the mean pH of their lumena was 7.05. These results indicate that the endocytic and autophagic pathways merge in the distal axon, resulting in a class of predegradative organelles that undergo regulated transport back to the cell body.

Central pattern generators (CPGs) are spinal neuronal networks required for locomotion. Glutamatergic neurons have been implicated as being important for intrinsic rhythm generation in the CPG and for the command signal for initiating locomotion, although this has not been demonstrated directly. We used a newly generated vesicular glutamate transporter 2-channelrhodopsin2-yellow fluorescent protein (Vglut2-ChR2-YFP) mouse to directly examine the functional role of glutamatergic neurons in rhythm generation and initiation of locomotion. This mouse line expressed ChR2-YFP in the spinal cord and hindbrain. ChR2-YFP was reliably expressed in Vglut2-positive cells and YFP-expressing cells could be activated by light. Photo-stimulation of either the lumbar spinal cord or the caudal hindbrain was sufficient to both initiate and maintain locomotor-like activity. Our results indicate that glutamatergic neurons in the spinal cord are critical for initiating or maintaining the rhythm and that activation of hindbrain areas containing the locomotor command regions is sufficient to directly activate the spinal locomotor network.

Females typically develop higher antibody responses and experience more adverse reactions following vaccination than males. These differences are observed in response to diverse vaccines, including the bacillus Calmette-Guerin vaccine, the measles, mumps and rubella vaccine, the yellow fever virus vaccine and influenza vaccines. Sex differences in the responses to vaccines are observed across diverse age groups, ranging from infants to aged individuals. Biological as well as behavioral differences between the sexes are likely to contribute to differences in the outcome of vaccination between the sexes. Immunological, hormonal, genetic and microbiota differences between males and females may also affect the outcome of vaccination. Identifying ways to reduce adverse reactions in females and increase immune responses in males will be necessary to adequately protect both sexes against infectious diseases.

Female mosquitoes are major vectors of human disease and the most dangerous are those that preferentially bite humans. A 'domestic' form of the mosquito Aedes aegypti has evolved to specialize in biting humans and is the main worldwide vector of dengue, yellow fever, and chikungunya viruses. The domestic form coexists with an ancestral, 'forest' form that prefers to bite non-human animals and is found along the coast of Kenya. We collected the two forms, established laboratory colonies, and document striking divergence in preference for human versus non-human animal odour. We further show that the evolution of preference for human odour in domestic mosquitoes is tightly linked to increases in the expression and ligand-sensitivity of the odorant receptor AaegOr4, which we found recognizes a compound present at high levels in human odour. Our results provide a rare example of a gene contributing to behavioural evolution and provide insight into how disease-vectoring mosquitoes came to specialize on humans.

Vitamin A and retinene, the carotenoid precursors of rhodopsin, occur in a variety of molecular shapes, cis-trans isomers of one another. For the synthesis of rhodopsin a specific cis isomer of vitamin A is needed. Ordinary crystalline vitamin A, as also the commercial synthetic product, both primarily all-trans, are ineffective. The main site of isomer specificity is the coupling of retinene with opsin. It is this reaction that requires a specific cis isomer of retinene. The oxidation of vitamin A to retinene by the alcohol dehydrogenase-cozymase system displays only a low degree of isomer specificity. Five isomers of retinene have been isolated in crystalline condition: all-trans; three apparently mono-cis forms, neoretinenes a and b and isoretinene a; and one apparently di-cis isomer, isoretinene b. Neoretinenes a and b were first isolated in our laboratory, and isoretinenes a and b in the Organic Research Laboratory of Distillation Products Industries. Each of these substances is converted to an equilibrium mixture of stereoisomers on simple exposure to light. For this reaction, light is required which retinene can absorb; i.e., blue, violet, or ultraviolet light. Yellow, orange, or red light has little effect. The single geometrical isomers of retinene must therefore be protected from low wave length radiation if their isomerization is to be avoided. By incubation with opsin in the dark, the capacity of each of the retinene isomers to synthesize rhodopsin was examined. All-trans retinene and neoretinene a are inactive. Neoretinene b yields rhodopsin indistinguishable from that extracted from the dark-adapted retina (lambda(max.) 500 mmicro). Isoretinene a yields a similar light-sensitive pigment, isorhodopsin, the absorption spectrum of which is displaced toward shorter wave lengths (lambda(max.) 487 mmicro). Isoretinene b appears to be inactive, but isomerizes preferentially to isoretinene a, which in the presence of opsin is removed to form isorhodopsin before the isomerization can go further. The synthesis of rhodopsin in solution follows the course of a bimolecular reaction, as though one molecule of neoretinene b combines with one of opsin. The synthesis of isorhodopsin displays similar kinetics. The bleaching of rhodopsin, whether by chemical means or by exposure to yellow or orange (i.e., non-isomerizing) light, yields primarily or exclusively all-trans retinene. The same appears to be true of isorhodopsin. The process of bleaching is therefore intrinsically irreversible. The all-trans retinene which results must be isomerized to active configurations before rhodopsin or isorhodopsin can be regenerated. A cycle of isomerization is therefore an integral part of the rhodopsin system. The all-trans retinene which emerges from the bleaching of rhodopsin must be isomerized to neoretinene b before it can go back; or if first reduced to all-trans vitamin A, this must be isomerized to neovitamin Ab before it can regenerate rhodopsin. The retina obtains new supplies of the neo-b isomer: (a) by the isomerization of all-trans retinene in the eye by blue or violet light; (b) by exchanging all-trans vitamin A for new neovitamin Ab from the blood circulation; and (c) the eye tissues may contain enzymes which catalyze the isomerization of retinene and vitamin A in situ. When the all-trans retinene which results from bleaching rhodopsin in orange or yellow light is exposed to blue or violet light, its isomerization is accompanied by a fall in extinction and a shift of absorption spectrum about 5 mmicro toward shorter wave lengths. This is a second photochemical step in the bleaching of rhodopsin. It converts the inactive, all-trans isomer of retinene into a mixture of isomers, from which mixtures of rhodopsin and isorhodopsin can be regenerated. Isorhodopsin, however, is an artefact. There is no evidence that it occurs in the retina; nor has isovitamin Aa or b yet been identified in vivo. In rhodopsin and isorhodopsin, the prosthetic groups appear to retain the cis configurations characteristic of their retinene precursors. In accord with this view, the beta-bands in the absorption spectra of both pigments appear to be cis peaks. The conversion to the all-trans configuration occurs during the process of bleaching. The possibility is discussed that rhodopsin may represent a halochromic complex of a retinyl ion with opsin. The increased resonance associated with the ionic state of retinene might then be responsible both for the color of rhodopsin and for the tendency of retinene to assume the all-trans configuration on its release from the complex. A distinction must be made between the immediate precursor of rhodopsin, neovitamin Ab, and the vitamin A which must be fed in order that rhodopsin be synthesized in vivo. Since vitamin A isomerizes in the body, it is probable that any geometrical isomer can fulfill all the nutritional needs for this vitamin.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

The properties of neurones of the guinea-pig deep cerebellar nuclei in a slice preparation were investigated by intracellular recording. The recorded population of cells did not differ morphologically from nuclear cells in vivo as judged from neurones stained with Lucifer Yellow. Fifty-two out of sixty cells were spontaneously active with a regular firing pattern and a mean frequency of 26 +/- 14 (mean +/- S.D.) impulses/s. The action potentials lasted 0.41 +/- 0.07 ms (n = 60) with an amplitude of 58 +/- 8 mV. Input resistance was 44 +/- 10 M omega and the time constant of the membrane 13 +/- 3 ms. When stimulated with intracellularly injected depolarizing current pulses the cells responded with trains of action potentials. Near the threshold for the spike the stimulation produced firing of constant frequency and from more hyperpolarized levels an initial acceleration sometimes followed by a deceleration was seen. At levels less than 15 mV from the spike threshold there was a rebound train of spikes as a response to a hyperpolarizing current injection. At more hyperpolarized levels there was only a small depolarizing potential after the hyperpolarizing stimulation. Three types of subthreshold potentials were recorded. Spikelets rose from base line as 3-10 mV depolarizing wavelets with a duration between 5 and 10 ms. They served as trigger potentials for the action potential. Plateau potentials were slow depolarizing potentials often reaching the spike threshold and thus generating long trains of action potentials. After-hyperpolarizations followed each spike with a time course dependent on the previous activity of the cell. Plots of the firing frequency versus injected current were linear at the first and second interspike interval, after 50 ms of activity and at steady state. Plots of the voltage versus injected current were upward concave demonstrating anomalous rectification of the cell membrane. It is concluded that neurones in the deep cerebellar nuclei in vitro are spontaneously active because of the electroresponsive properties of their membranes. The physiological importance may be that the cerebellar output from these cells can be rapidly and efficiently modulated by synaptic potentials generated by Purkinje cells and mossy and climbing fibres.

The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Galpha and Gbetagamma-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose-response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of alpha-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose-response curve and the downstream dose-response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast.

Fura-2 recording of Ca2+ influx was used to show that incubation in 1 microm nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHbetaE-sensitive component that presumably corresponds to alpha4beta2 receptors. To study changes in alpha4beta2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the alpha4 or beta2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca2+ influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent alpha4 and beta2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of alpha4beta2 receptors, coexpression of the alpha4-YFP and beta2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic alpha4beta2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 microm nicotine, there was increased FRET in the cell body, denoting increased assembly of alpha4beta2 receptors. Thus, changes in alpha4beta2 receptor assembly play a role in the regulation of alpha4beta2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca2+-stimulated pathways.