A study of photoreceptor-retinal pigment epithelium complex: age-related changes in monkeys

Retinas of 4-, 10-, and 20-year-old monkeys were studied by light microscopy, electron microscopy, and scanning electron microscopy. Sections from the midperipheral region of every retina were selected for comparison. Although no significant differences were found between 4- and 10-year-old retinas, four major changes were found in 20-year-old monkey retinas: (i) increased number of displaced photoreceptor cells (DPC), (ii) increased number of macrophages of different morphology in subretinal space, (iii) increase in pigment granules in retinal pigment epithelium (RPE) cells, and (iv) altered morphology of Muller cells. DPC included both rods and cones. Their location and morphology depended on the stage of their displacement. These cells were usually oval or rounded in shape and were found either among the outer segments of other photoreceptor cells, having stalks extending into the outer nuclear layer, or were located in the subretinal space and had no stalk. A narrow space around the DPC stalks, indicating a change in the intercellular connection between photoreceptor cells and Muller cells, was observed. Furthermore, the Muller cells related to DPC had shortened and markedly reduced microvilli. Two types of macrophages were found in the subretinal space of aged monkey retinas. One type was similar in morphology to RPE cells. Some of these cells were noticed detaching from RPE. Other types of macrophages were nonpigmented. The modifications in RPE were closely related to the changes in the associated neuroretina. The RPE cells in aged retina were devoid of microvilli or had a few thin microvilli. The pleomorphic pigment granules were dispersed throughout the cytoplasm. These cells varied in their size, shape, and surface features. These changes could significantly alter the retinal metabolic equilibrium and may be indicative of age related degenerative processes.     

Inhibition of protein synthesis enhances transformation of primary cells by viral DNA

Inhibition of protein synthesis by cycloheximide after transfection and subsequent removal of the drug increased the transformation efficiency of primary cells by plasmids containing the left 4.5, 6.7, or 16% of the adenovirus (Ad) genome. The enhancement factor ranged from 2 to as much as 70 depending on the size of the viral DNA fragments used. Addition of cycloheximide before or at the time of transfection inhibited transformation, suggesting that viral protein synthesis is important during the early phase of transformation. Transient expression assays showed that cells treated with cycloheximide post-transfection contained as much as three times the amount of viral RNA transcribed from regions E1A and E1B. Conversion of a rat cell line lacking thymidine kinase activity (TK-) to the TK+ phenotype by a plasmid containing the herpes TK gene was severely inhibited by the drug treatment, suggesting that the enhancement effects of cycloheximide on transformation may be specific for Ad DNA. Cycloheximide treatment also increased the number of transformants induced by a transformation defective E1B mutant of Ad12 (cyt mutant). Plasmid containing only the E1A region of Ad12 transformed primary rat kidney cells with very low efficiency. The inclusion of E1B in the transfecting DNA fragments increased the transformation frequency by more than 400-fold, much higher than that achieved by cycloheximide. Thus, cycloheximide cannot replace E1B functions in transformation efficiency.     

Hypotensive and vasodilatory activity of (+/-) 1-o-octadecyl-2-acetyl glyceryl-3-phosphorylcholine in the normotensive rat

Synthetic (+/-) 1-O-octadecyl-2-acetyl-glyceryl-3-phosphorylcholine (octadecyl-AGPC) in microgram/kg doses given intravenously effectively and potently lowered mean arterial blood pressure in conscious and anesthetized normotensive rats. The hypotensive activity was much more pronounced in the anesthetized rat than in the conscious rat. The hypotension was associated with a significant elevation in plasma renin activity (PRA). In the rat in which the hindquarters were perfused, octadecyl-AGPC given intraarterially effectively decreased the perfusion and systemic pressures in a dose-dependent manner. Pharmacological blockade with specific cholinergic, histaminergic or beta-adrenergic receptor antagonists, did not block or attenuate the octadecyl-AGPC-induced reduction in perfusion or systemic pressure. These results suggest that the hypotensive activity of octadecyl-AGPC in the normotensive rat is the result of direct vasodilation and not the result of cholinergic, histaminergic or beta-adrenergic receptor interaction.     

Some factors affecting spore replication of Nosema algerae (Microspora,Nosematidae) in Pieris brassicae (Lepidoptera)

The production of Nosema algerae spores was examined in Pieris brassicae. Spore replication in the insect host followed a logistic pattern of development. The factors studied which affected spore production and replication were dose level (5 × 10², 5 × 10³, and 5 × 10⁴ spores per insect), larval instar (fourth and fifth), and cool pretreatment of the insects at 20°C prior to inoculation compared with a constant temperature of 26°C. A three-way analysis showed the interactions between these factors. The logistic pattern of spore replication was used to explain the results.     

Comparison of ultrafiltration systems for concentration of biologicals

Ultrafiltration has been used with increasing frequency in recent years in biological laboratories for concentration, separation or purification of biological material. No data have been available on the comparison of the characteristics of Ultrafiltration systems currently in use. This study compares the filtration characteristics of four systems using commercially available membranes on suspensions and solutions: suspension of protein micelles (casein), cell debris (E. coli) and catalase solution. None of the four systems considered is found to be generally superior for all the suspensions and solutions. Vibration systems were most effective when relatively large particles were involved, while laminar flow recycling systems with high wall shear rates were best for dilute suspensions and proteins in solution. It was found that shearing inactivates enzymes in both recycle and vibration systems. It was also observed that vibration actually reduces flux in dilute solutions.     

Selective degradation of insulin within rat liver endosomes

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.     

Cleavage of dengue virus NS1-NS2A requires an octapeptide sequence at the C terminus of NS1.

The length of amino acid sequence at the NS1-NS2A juncture of dengue virus that is required for specific cleavage effected by the cis-acting function of NS2A was identified by deletion analysis. Recombinant DNA sequences of NS1-NS2A, each containing a deletion in NS1 followed by a sequence of 3 to 20 amino acids at the C terminus of NS1 preceding the cleavage site, were constructed and expressed with vaccinia virus as a vector. The NS1 product of recombinant vaccinia virus-infected cells was immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The occurrence of cleavage between NS1 and NS2A was indicated by the appearance of shortened NS1. Failure to cleave this site yielded a large NS1-NS2A fusion protein. This analysis indicated that a minimum length of eight amino acids at the NS1 C terminus preceding the NS1-NS2A juncture is required for cleavage to take place. Comparison of this eight-amino-acid sequence of the NS1 C terminus of dengue type 4 virus with the analogous sequences of 12 other flaviviruses suggests that the consensus cleavage site sequence is as follows: (table; see text)     

Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes

Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type 4 genes. These results demonstrate that both dengue virus structural and nonstructural proteins are targets for the cytotoxic T-cell-mediated immune response to dengue virus and suggest that serotype-cross-reactive CD8+ CTL may be important mediators of viral clearance and of virus-induced immunopathology during secondary dengue virus infections.