A perceptual memory for low-contrast visual signals

Detection of a visual signal can be facilitated by simultaneous presentation of a similar subthreshold signal. Here we show that the facilitatory effect of a subthreshold signal can persist for more than 16 s. Presenting a near-threshold Gabor signal (prime) produced a phase-independent increase in contrast sensitivity (40%) to similar successive signals (target) for a period of up to 16 s. This effect was obtained only when both prime and target were presented to the same eye. We further show that the memory trace is inactivated by presenting high-contrast signals before the target. These results suggest that activated neurons in the primary visual cortex retain a near-threshold memory trace that persists until reactivated.

Excitatory–inhibitory network in the visual cortex: Psychophysical evidence

At early stages in visual processing cells respond to local stimuli with specific features such as orientation and spatial frequency. Although the receptive fields of these cells have been thought to be local and independent, recent physiological and psychophysical evidence has accumulated, indicating that the cells participate in a rich network of local connections. Thus, these local processing units can integrate information over much larger parts of the visual field; the pattern of their response to a stimulus apparently depends on the context presented. To explore the pattern of lateral interactions in human visual cortex under different context conditions we used a novel chain lateral masking detection paradigm, in which human observers performed a detection task in the presence of different length chains of high-contrast-flanked Gabor signals. The results indicated a nonmonotonic relation of the detection threshold with the number of flankers. Remote flankers had a stronger effect on target detection when the space between them was filled with other flankers, indicating that the detection threshold is caused by dynamics of large neuronal populations in the neocortex, with a major interplay between excitation and inhibition. We considered a model of the primary visual cortex as a network consisting of excitatory and inhibitory cell populations, with both short- and long-range interactions. The model exhibited a behavior similar to the experimental results throughout a range of parameters. Experimental and modeling results indicated that long-range connections play an important role in visual perception, possibly mediating the effects of context.

Perceptual motion standstill in rapidly moving chromatic displays

In motion standstill, a quickly moving object appears to stand still, and its details are clearly visible. It is proposed that motion standstill can occur when the spatiotemporal resolution of the shape and color systems exceeds that of the motion systems. For moving red-green gratings, the first- and second-order motion systems fail when the grating is isoluminant. The third-order motion system fails when the green/red saturation ratio produces isosalience (equal distinctiveness of red and green). When a variety of high-contrast red-green gratings, with different spatial frequencies and speeds, were made isoluminant and isosalient, the perception of motion standstill was so complete that motion direction judgments were at chance levels. Speed ratings also indicated that, within a narrow range of luminance contrasts and green/red saturation ratios, moving stimuli were perceived as absolutely motionless. The results provide further evidence that isoluminant color motion is perceived only by the third-order motion system, and they have profound implications for the nature of shape and color perception.

Patterning of functional antibodies and other proteins by photolithography of silane monolayers.

We have demonstrated the assembly of two-dimensional patterns of functional antibodies on a surface. In particular, we have selectively adsorbed micrometer-scale regions of biotinylated immunoglobulin that exhibit specific antigen binding after adsorption. The advantage of this technique is its potential adaptability to adsorbing arbitrary proteins in tightly packed monolayers while retaining functionality. The procedure begins with the formation of a self-assembled monolayer of n-octadecyltrimethoxysilane (OTMS) on a silicon dioxide surface. This monolayer can then be selectively removed by UV photolithography. Under appropriate solution conditions, the OTMS regions will adsorb a monolayer of bovine serum albumin (BSA), while the silicon dioxide regions where the OTMS has been removed by UV light will adsorb less than 2% of a monolayer, thus creating high contrast patterned adsorption of BSA. The attachment of the molecule biotin to the BSA allows the pattern to be replicated in a layer of streptavidin, which bonds to the biotinylated BSA and in turn will bond an additional layer of an arbitrary biotinylated protein. In our test case, functionality of the biotinylated goat antibodies raised against mouse immunoglobulin was demonstrated by the specific binding of fluorescently labeled mouse IgG.

Rethinking cell structure.

Cell structure, emerging from behind the veil of conventional electron microscopy, appears far more complex than formerly realized. The standard plastic-embedded, ultrathin section can image only what is on the section surface and masks the elaborate networks of the cytoplasm and nucleus. Embedment-free electron microscopy gives clear, high-contrast micrographs of cell structure when combined with removal of obscuring material such as soluble proteins. The resinless ultrathin section is the technique of choice; it is simple and inexpensive, and it uses ordinary electron microscopes. The resulting pictures reveal a world of complex cell structure and function. These images necessarily change our conception of the cytoskeleton, nuclear matrix, mitosis, and the relation of membranes to cytostructure.

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis

Physiological studies with excised stem segments have implicated the plant hormone indole-3-acetic acid (IAA or auxin) in the regulation of cell elongation. Supporting evidence from intact plants has been somewhat more difficult to obtain, however. Here, we report the identification and characterization of an auxin-mediated cell elongation growth response in Arabidopsis thaliana. When grown in the light at high temperature (29°C), Arabidopsis seedlings exhibit dramatic hypocotyl elongation compared with seedlings grown at 20°C. This temperature-dependent growth response is sharply reduced by mutations in the auxin response or transport pathways and in seedlings containing reduced levels of free IAA. In contrast, mutants deficient in gibberellin and abscisic acid biosynthesis or in ethylene response are unaffected. Furthermore, we detect a corresponding increase in the level of free IAA in seedlings grown at high temperature, suggesting that temperature regulates auxin synthesis or catabolism to mediate this growth response. Consistent with this possibility, high temperature also stimulates other auxin-mediated processes including auxin-inducible gene expression. Based on these results, we propose that growth at high temperature promotes an increase in auxin levels resulting in increased hypocotyl elongation. These results strongly support the contention that endogenous auxin promotes cell elongation in intact plants.

High-precision 40Ar/39Ar geochronology and the advent of North America’s Late Cretaceous terrestrial fauna

A densely sampled, diverse new fauna from the uppermost Cedar Mountain Formation, Utah, indicates that the basic pattern of faunal composition for the Late Cretaceous of North America was already established by the Albian-Cenomanian boundary. Multiple, concordant 40Ar/39Ar determinations from a volcanic ash associated with the fauna have an average age of 98.39 ± 0.07 million years. The fauna of the Cedar Mountain Formation records the first global appearance of hadrosaurid dinosaurs, advanced lizard (e.g., Helodermatidae), and mammal (e.g., Marsupialia) groups, and the first North American appearance of other taxa such as tyrannosaurids, pachycephalosaurs, and snakes. Although the origin of many groups is unclear, combined biostratigraphic and phylogenetic evidence suggests an Old World, specifically Asian, origin for some of the taxa, an hypothesis that is consistent with existing evidence from tectonics and marine invertebrates. Large-bodied herbivores are mainly represented by low-level browsers, ornithopod dinosaurs, whose radiations have been hypothesized to be related to the initial diversification of angiosperm plants. Diversity at the largest body sizes (>106 g) is low, in contrast to both preceding and succeeding faunas; sauropods, which underwent demise in the Northern hemisphere coincident with the radiation of angiosperms, apparently went temporarily unreplaced by other megaherbivores. Morphologic and taxonomic diversity among small, omnivorous mammals, multituberculates, is also low. A later apparent increase in diversity occurred during the Campanian, coincident with the appearance of major fruit types among angiosperms, suggesting the possibility of adaptive response to new resources.

Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR(-)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50°C. The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2. Transgenic potato plants (cultivar Desirée) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation. In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance against PSTVd replication was stably inherited to the vegetative progenies.

Low- and high-level transgenic expression of β2-adrenergic receptors differentially affect cardiac hypertrophy and function in Gαq-overexpressing mice

Transgenic overexpression of Gαq in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed β-adrenergic receptor (βAR) function. The role of βAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the βAR kinase (βARK), which acts to inhibit the kinase, or concomitant overexpression of the β2AR at low (≈30-fold, Gαq/β2ARL), moderate (≈140-fold, Gαq/β2ARM), and high (≈1,000-fold, Gαq/β2ARH) levels above background βAR density. Expression of the βARK inhibitor had no effect on the phenotype, consistent with the lack of increased βARK levels in Gαq mice. In marked contrast, Gαq/β2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and α-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized β2AR/AC signal cannot be excluded. Higher expression of receptors in Gαq/β2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With ≈1,000-fold overexpression, the majority of Gαq/β2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of βAR signaling is deleterious in heart failure, selective enhancement by overexpressing the β2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.

Maintenance of Epstein–Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP, enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1–89 and 323–386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379–641), which includes the nuclear localization signal and DNA-binding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP-containing plasmid DNA in transient replication assays. The importance of chromosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1–378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-I amino acids 1–90 or histone H1–2 could substitute for EBNA-1 amino acids 1–378 in mediating more efficient accumulation of replicated oriP plasmid, association with mitotic chromosomes, nuclear retention, and long-term episome persistence. These data strongly support the hypothesis that mitotic chromosome association is a critical factor for episome maintenance. The replacement of 60% of EBNA-1 with cell protein is a significant step toward eliminating the need for noncellular protein sequences in the maintenance of episomal DNA in human cells.

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein

The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT) the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent such as EDTA. The resultant difference in chemical reactivity provides a means to measure T in the absence of EDTA, (MT + T) in its presence, and, of course, MT by difference. The 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide derivative of T can be isolated from tissue homogenates by HPLC and quantified fluorimetrically with a detection limit in the femtomolar range and a linear response over 3 orders of magnitude. Analysis of liver, kidney, and brain of rats reveals almost as much T as MT. Moreover, in contrast to earlier views, MT in tissue extracts appears to be less stable than T. The existence of T in tissues under normal physiological conditions has important implications for its function both in zinc metabolism and the redox balance of the cell.

Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activity in vivo

The integrin αLβ2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the αM and α2 subunits has been crystallized in both open and closed conformations; however, the αL I domain has been crystallized in only the closed conformation. We hypothesized that the αL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the αL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg2+. Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The kon, koff, and KD values for the locked open I domain were within 1.5-fold of values previously determined for the αLβ2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized αLβ2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.

The high spontaneous mutation rate: Is it a health risk?*

The human mutation rate for base substitutions is much higher in males than in females and increases with paternal age. This effect is mainly, if not entirely, due to the large number of cell divisions in the male germ line. The mutation-rate increase is considerably greater than expected if the mutation rate were simply proportional to the number of cell divisions. In contrast, those mutations that are small deletions or rearrangements do not show the paternal age effect. The observed increase with the age of the father in the incidence of children with different dominant mutations is variable, presumably the result of different mixtures of base substitutions and deletions. In Drosophila, the rate of mutations causing minor deleterious effects is estimated to be about one new mutation per zygote. Because of a larger number of genes and a much larger amount of DNA, the human rate is presumably higher. Recently, the Drosophila data have been reanalyzed and the mutation-rate estimate questioned, but I believe that the totality of evidence supports the original conclusion. The most reasonable way in which a species can cope with a high mutation rate is by quasi-truncation selection, whereby a number of mutant genes are eliminated by one “genetic death.”