Light-induced increases in cGMP metabolic flux correspond with electrical responses of photoreceptors

The metabolism of photoreceptor cGMP and the relationship of its light-sensitive regulation to rhodopsin photoisomerization and to the photoreceptor electrical response was examined in isolated, intact rabbit retinas. The dynamics of cGMP metabolism were assessed by measuring the rate of 18O incorporation from 18O-water into the alpha-phosphoryls of the guanine nucleotides. The photoreceptor electrical response was determined by measuring the aspartate-isolated mass receptor potential. Basal cGMP flux in dark-adapted retinas was 33 pmol cGMP X mg protein-1 X s-1 which translates into a metabolic rate in the rod outer segment (ROS) of 1.7 mM/min in ATP equivalents. Photic stimulation increased this flux as much as 4.5-fold. With continuous illumination, increasing intensity caused increments in cGMP metabolic flux to a maximum of 4.5-fold, with corresponding increases in the electrical response over the same 3-log unit intensity range. Tight coupling between activation of guanylate cyclase and phosphodiesterase was indicated by either no changes in cGMP steady state concentrations or relatively small fluctuations represented by increases of 50% at lower light intensities and a 12% decrease at one of the highest intensities. A stoichiometry of about 10,000 molecules of cGMP generated and hydrolyzed per photon absorbed was calculated for the lowest light intensity when the increment in cGMP metabolic flux per photon was maximal. Flashing light caused an increase in flux in proportion to frequency up to 1 Hz and a nearly proportional increase in the voltage time integral of the electrical response up to 0.5 Hz. This indicates that the temporal resolution, or "on"/"off" rate, of the cGMP metabolic response was as fast or faster than the temporal resolution of the electrical response. The concentration of cGMP remained relatively stable in spite of the marked acceleration of cGMP flux that occurred over the 32-fold range of frequencies tested. Taken together these results show that the light-accelerated rate of cGMP synthesis tightly coupled to hydrolysis becomes a primary energy-utilizing system in the photoreceptor and represents a response that fulfills certain of the fundamental criteria required of a metabolic event playing an essential role in phototransduction.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

Simultaneous induction of antigen-specific IgA helper T cells and IgG suppressor T cells in the murine Peyer's patch after protein feeding

The effects of feeding the dietary protein antigen, ovalbumin (OVA), on OVA-specific IgG and IgA immune responses involving Peyer's patches (PP) and mesenteric lymph nodes (MLN) were examined. Mice were administered soluble OVA by gastric intubation. One to 3 days later, PP, MLN, or spleen cells from these donor mice were adoptively transferred into normal syngeneic recipients. After two subsequent immunizations, spleens from the recipient mice were assayed for IgA and IgG anti-OVA plaque-forming cell (PFC) responses. None of the tissues from normal (unfed) mice had the inherent ability to alter recipients' IgG or IgA PFC responses. Within 1 day of OVA feeding, however, cells were generated in the PP that could augment recipients' IgA anti-OVA PFC responses and suppress IgG PFC responses. Three days after OVA feeding, these cells were present in MLN as well, and whereas the IgG suppressor cell also appeared to migrate to spleen, the IgA helper cell did not. The cells mediating antigen-specific IgG suppression and IgA help were both T cells but could be distinguished by surface phenotype. We therefore conclude that protein feeding induces differential, isotype-specific immunoregulation in gut-associated lymphoid tissues, part of which is mediated by an antigen-specific IgA helper T cell.     

Immunoregulatory function of T cells activated in the autologous mixed lymphocyte reaction

This study was undertaken to define the functional properties of T cells stimulated in the autologous mixed lymphocyte reaction (MLR) by purified B cells or macrophages. In preliminary experiments, it was found that T cells that had been cultured with autologous non-T cells inhibited pokeweed mitogen- (PWM) stimulated immunoglobulin synthesis by autologous B cells. In addition, the T cell-mediated suppression was eliminated by x-irradiation and hydrocortisone treatment, was mediated by a mechanism that occurred early in the PWM-stimulated cultures, and did not involve killing of mature immunoglobulin-secreting cells. T cells were then cultured with either autologous B cells or macrophages in order to determine whether such autoreactive T cells had a similar capacity to regulate PWM-induced immunoglobulin synthesis. Although T cell populations stimulated either by B cells or by macrophages suppressed proliferative responses and immunoglobulin synthesis, both these populations of autoreactive T cells provided help for immunoglobulin synthesis that was not significantly different from that provided by fresh T cells. These results suggest that the predominant functional consequence of activation of T cells in the autologous MLR is the generation of suppressor T cells capable of inhibiting immunoglobulin synthesis. Thus, the autologous MLR may represent a negative feedback mechanism for the regulation of the immune response.     

Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.     

Evidence for a causal role of CD38 expression in granulocytic differentiation of human HL-60 cells

Granulocytic differentiation of human HL-60 cells can be induced by retinoic acid and is accompanied by a massive expression of CD38, a multi-functional enzyme responsible for metabolizing cyclic ADP-ribose (cADPR), a Ca(2+) messenger. Immunofluorescence staining showed that CD38 was expressed not only on the surface of intact HL-60 cells but also intracellularly, which was revealed after permeabilization with Triton. Concomitant with CD38 expression was the accumulation of cADPR, and both time courses preceded the onset of differentiation, suggesting a causal role for CD38. Consistently, treatment of HL-60 cells with a permeant inhibitor of CD38, nicotinamide, inhibited both the CD38 activity and differentiation. More specific blockage of CD38 expression was achieved by using morpholino antisense oligonucleotides targeting its mRNA, which produced a corresponding inhibition of differentiation as well. Similar inhibitory effects were observed when CD38 expression was reduced by the RNA interference technique targeting two separate regions of the coding sequence of CD38. Further support came from transfecting HL-60 cells with a Tet-On expression vector containing a full-length CD38. Subsequent treatments with doxycycline induced both CD38 expression and differentiation in the absence of retinoic acid. These results provide the first evidence that CD38 expression and the consequential accumulation of cADPR play a causal role in mediating cellular differentiation.