Absence of linear subthreshold summation between red-green and luminance mechanisms over a wide range of spatio-temporal conditions

We have tested the independence of red-green chromatic and luminance mechanisms at detection threshold using a method of subthreshold summation. Stimuli were isoluminant red-green gratings and yellow-black luminance gratings that uniquely activate the red-green color and luminance mechanisms, respectively. Stimuli were Gaussian enveloped 0.25, 1 or 4 cpd sinewave gratings, counter-phase flickered at 0, 5 or 9 Hz. The threshold detection of red-green color contrast was measured in the presence of a subthreshold amount of luminance contrast, and vice versa. The results allow a model of linear summation between the color and luminance mechanisms to be rejected, but are well fitted by a model, assuming that these mechanisms are independent but combine to determine detection by probability summation, with a high summation index (median value = 4). We conclude that there are independent red-green chromatic mechanism and luminance detection mechanisms over this range of spatio-temporal conditions.     

Postreceptoral chromatic detection mechanisms revealed by noise masking in three-dimensional cone contrast space

We used a noise masking technique to test the hypothesis that detection is subserved by only two chromatic postreceptoral mechanisms (red-green and blue-yellow) and one achromatic (luminance) mechanism. The task was to detect a 1-c/deg Gaussian enveloped grating presented in a mask of static, spatially low-passed binary or Gaussian distributed noise. In the main experiment, the direction of the test stimulus (termed the signal) was constant in cone contrast space, and the direction of the noise was sampled in equally spaced directions within a plane (the noise plane) in the space. The signal was chosen to coincide with one of the three cardinal directions of three postulated mechanisms. The noise plane was selected to span two of the cardinal directions, including that chosen as the signal direction. As the noise direction was sampled around the noise plane, the signal detection threshold was found to vary in accordance with a linear cosine model, which predicted noise directions yielding maximum and minimum masking of the signal. In the direction of minimum masking (termed a null direction), the noise was found to have no masking effect on the signal. Moreover, the null was not orthogonal to the signal direction but lay instead in one of the cardinal directions. Our findings suggest that detection is mediated by only three mechanisms. In a further experiment we found little or no cross masking between each pair of cardinal directions up to the limit of our noise mask contrasts. This further supports the presence of no more than three independent postreceptoral mechanisms.     

Extended aromatic furan amidino derivatives as anti-Pneumocystis carinii agents

The syntheses of nine new derivatives of 2, 5-bis[4-(N-alkylamidino)phenyl]furans with extended aromatic systems are reported. The interaction of these dicationic furans with poly(dA)poly(dT) and with the duplex oligomers d(CGCGAATTCGCG)2 and d(GCGAATTCGC)2 was determined by Tm measurement, and the effectiveness of these compounds against the immunosuppressed rat model of Pneumocystis carinii was evaluated. At a screening dose of 10 micromol/kg, 4 of the 12 amidino furans described here are more active than the parent compound 1. In general, extension of the aromatic system in the absence of a substitution of the amidino nitrogens resulted in higher affinity for DNA than the parent compound as judged by the larger DeltaTm values and suggests enhanced van der Waals interactions in the amidino furan-DNA complex. Three of the compounds, 3, 5, and 11, yield cysts counts of less than 0.1% of control when administered at a dosage of 10 micromol/kg. Compound 3, which does not have an extended aromatic system, is the most active derivative. Although a direct correlation between anti-P. carinii activity and DNA binding affinity was not observed, all compounds which have significant activity have large DeltaTm values.     

The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse

Mouse sperm-egg fusion was examined using two-photon and confocal microscopy. A delay of several minutes occurred between the first observable event of fusion (which was the diffusion of Ca2+-sensitive dyes from egg into sperm) and any change in egg cytoplasmic Ca2+. When indo-1 dextran was used to obtain ratiometric two-photon images, there was no detectable local increase in egg cytoplasmic Ca2+ near the site of sperm fusion. However, the sperm underwent a Ca2+ transient which appeared to be coincident with the egg cytoplasm Ca2+ transient, which suggested that there was a high permeability pathway for Ca2+ between egg and sperm. To exclude this pathway from providing trigger Ca2+ for the egg transient, we reduced bathing [Ca2+] to approx. 18 microM and 13nM (with EGTA). In these conditions the first egg Ca2+ transient was not prevented, which makes an obligatory role for extracellular Ca2+ in the initiation of the egg Ca2+ transient unlikely. Both FITC-albumin (70 kDa) and 10 kDa dextran-linked Ca2+ indicators were able to diffuse into the sperm from the egg. In addition, phycoerythrin (240 kDa) rapidly diffused into the sperm shortly after fusion (but before any changes in Ca2+ occurred). This suggests that the 'pore(s)' that form during sperm-egg fusion must be at least 8 nm in diameter. These data are compatible with the idea that a diffusible sperm protein could trigger the observed changes in intracellular Ca2+ in the egg, but do not exclude the possibility that other second messengers are generated during sperm-egg fusion.     

Expansion of autoreactive T cells in multiple sclerosis is independent of exogenous B7 costimulation

Multiple sclerosis (MS) is an inflammatory disease of the myelinated central nervous system that is postulated to be induced by myelin-reactive CD4 T cells. T cell activation requires an antigen-specific signal through the TCR and a costimulatory signal, which can be mediated by B7-1 or B7-2 engagement of CD28. To directly examine the activation state of myelin-reactive T cells in MS, the costimulation requirements necessary to activate myelin basic protein (MBP) or tetanus toxoid (TT)-reactive CD4 T cells were compared between normal controls and MS patients. Peripheral blood T cells were stimulated with Chinese hamster ovary (CHO) cells transfected either with DRB1*1501/DRA0101 chains (t-DR2) alone, or in combination with, B7-1 or B7-2. In the absence of costimulation, T cells from normal subjects stimulated with the recall antigen TT p830-843 were induced to expand and proliferate, but stimulation with MBP p85-99 did not have this effect. In marked contrast, T cells from patients with MS stimulated with MBP p85-99 in the absence of B7-1 or B7-2 signals expanded and proliferated. Thus, MBP-reactive CD4 T cells in patients with MS are costimulation independent and have been previously activated in vivo. These experiments provide further direct evidence for a role of activated MBP-specific CD4 T cells in the pathogenesis of MS.     

Syntheses of D- and L-myo-inositol 1,2,4,5-tetrakisphosphate and stereoselectivity of the I(1,4,5)P3 receptor binding

D- and L-myo-Inositol 1,2,4,5-tetrakisphosphate [D- & L-I(1,2,4,5)P4], which are analogues of D-myo-Inositol 1,4,5-trisphosphate [D-I(1,4,5)P3], a calcium mobilizing second messenger, were synthesized via resolution of the camphanate ester of a myo-inositol derivative, and the binding affinities to I(1,4,5)P3 receptor were measured.     

Identification and analysis of genes involved in anaerobic toluene metabolism by strain T1: putative role of a glycine free radical

The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. The tutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.     

Pharmacological characterization of PD 152255, a novel dimeric benzimidazole dopamine D3 antagonist

152255 (E-1,1'-(2-butene-1,4-diyl)bis[2-[4-[3-(1-piperidinyl)propoxy]-phe nyl]-1H-benzimidazole]) exhibited high affinity (Ki = 12.7 nM) for human dopamine (DA) D3 receptors expressed in CHO K1 cells but not for DA D2L receptors (Ki = 565 nM), DA D42 or DA D1 receptors (Ki > 3 microM) and a number of other neurotransmitter receptors. Affinity for human muscarinic receptors was seen in vitro but no functional muscarinic agonist and/or antagonist action was observed in vivo. Antagonist activity at DA D3 receptors was demonstrated by blockade of quinpirole-stimulated [3H]-thymidine uptake in D3 transfected cells, an effect that was 28-fold more potent than in D2-transfected cells. Unlike classical DA D2 antagonists, PD 152255 did not increase rat brain DA synthesis and it increased locomotion in habituated rats. However, like antipsychotics, PD 152255 reduced locomotor activity in mice and reduced spontaneous and amphetamine-stimulated locomotion in nonhabituated rats. These results demonstrate that PD 152255 is a DA D3 antagonist that may have antipsychotic activity.     

Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat

We previously reported (K. T. Jeang, R. Chun, N. H. Lin, A. Gatignol, C. G. Glabe, and H. Fan, J. Virol. 67: 6224-6233, 1993) that human immunodeficiency virus type 1 (HIV-1) Tat and Sp1 form a protein-protein complex. Here, we have characterized the physical interaction and a functional consequence of Tat-Sp1 contact. Using in vitro protein chromatography, we mapped the region in Tat that contacts Sp1 to amino acids 30 to 55. We found that in cell-free reactions, Tat augmented double-stranded DNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation in a contact-dependent manner. Tat mutants that do not bind Sp1 failed to influence phosphorylation of the latter. In complementary experiments, we also found that Tat forms protein-protein contacts with DNA-PK. We confirmed that in HeLa and Jurkat cells, Tat expression indeed increased the intracellular amount of phosphorylated Sp1 in a manner consistent with the results of cell-free assays. Furthermore, using two phosphatase inhibitors and a kinase inhibitor, we demonstrated a modulation of reporter gene expression as a consequence of changes in Sp1 phosphorylation. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1 which is affected by Tat and DNA-PK.