An eye-derived growth factor regulates epithelial cell proliferation in the cultured lens

Lenses in organ culture permit an analysis of factors acting on epithelial cell growth, while keeping the normal steric constraints of the cell population. By employing this technique with radioautography of epithelial whole mounts, we showed that the DNA synthesis found in the epithelia of cultured bovine lenses follows an organized spatial and temporal pattern during culture. Within the first 48 h, active cells were located at the preequatorial region ("germinative zone"), a distribution consistent with the in vivo spatial organization of multiplying cells. Starting at about 48 h, cells from the central region of the epithelium--a nonproliferating population--were triggered to synthesize DNA in the presence of eye-derived growth factor (EDGF). When cultured in serum-free medium, only a small fraction of the cells was labeled, but when a low serum concentration was present, this fraction reached 50% of the cell population. The stimulatory effect of EDGF required a lag period, but its effect reached a maximum exceeding that found for serum. However, the cells from the germinative region, having a cell density three- to four-fold higher than the central region, were not stimulated to proliferate. This occurred irrespective of the presence of EDGF or serum. If this growth-stimulatory activity derived from the retina were the actual factor controlling cell proliferation in the lens in vivo, then the results presented here would point to the presence of a regulatory mechanism similar to that known for some other hormones.     

Light effects in yeast: inhibition by visible light of growth and transport in Saccharomyces cerevisiae grown at low temperatures.

Growth rate, sugar transport, and amino acid transport of yeast cells grown at 12 degrees C were inhibited by cool-white fluorescent light. At light intensities below 1,250 lx, growth and membrane transport were only slightly inhibited. Above 1,250 lx, there was increasing inhibition of both processes. Transport of histidine was completely inhibited after 3 to 5 days in cultures grown at 12 degrees C under 3,500-lx illumination. Cells grown at 20 degrees C were not inhibited by light intensities that caused complete loss of viability and membrane transport activity in cells grown at 12 degrees C.     

Galactose transport in Saccharomyces cerevisiae. II. Characteristics of galactose uptake and exchange in galactokinaseless cells

The characteristics of the inducible galactose system in Saccharomyces cerevisiae were studied by using the nonmetabolized galactose analogues, l-arabinose and d-fucose, and galactokinaseless and transportless mutants. Induced wild-type cells transport l-arabinose by facilitated diffusion. Transportless cells transport neither galactose nor l-arabinose above the noninduced rate, whereas galactokinaseless cells transport galactose l-arabinose and d-fucose by facilitated diffusion. Determination of unidirectional rate of (14)C-labeled galactose uptake by preloaded galactokinaseless cells, containing a large unlabeled free-galactose pool, showed that the rate of galactose uptake by facilitated diffusion is greater than the rate of galactose metabolism at similar external galactose concentrations.     

Relationship between sugar structure and competition for the sugar transport system in Bakers' yeast

Twenty-five sugars have been compared as inhibitors of l-sorbose or d-xylose transport by the constitutive, monosaccharide transport system in bakers' yeast. d-Glucose showed the highest activity (i.e., apparent K(i) = 5 mm). Since all sugars except 2-deoxyglucose showed a decrease in activity relative to glucose (i.e., apparent K(i) = 25 - >2,000 mm), an attempt was made to relate the activity of each sugar with the way its structure differs from that of d-glucose. Assuming that the inhibition was the result of sugar-carrier complex formation, the analysis showed that the transport system has a rather broad specificity for pyranoses. Single changes at each of the five carbons of d-glucose (except for the 2-deoxy derivative) result in variable decreases in activity depending upon the carbon number and the alteration. The largest decrease in activity effected by a single change is the methylation or glucosylation of the anomeric hydroxyl. The combination of two or more changes leads to a decrease which is greater than the decrease in activity resulting from the individual changes occurring alone.     

Glucose transport in vesicles reconstituted from Saccharomyces cerevisiae membranes and liposomes.

Glucose transport activity was reconstituted into liposomes by the freeze-thaw-sonication procedure from unextracted Saccharomyces cerevisiae membranes and preformed phospholipid liposomes. Fluorescence-dequenching measurements with octadecylrhodamine B chloride (R18)-labeled membranes showed that the yeast membrane lipids are diluted by the liposome lipids after the freeze-thaw-sonication procedure. At lipid-to-protein ratios greater than 75:1, vesicles with single transporters were formed. Reconstituted specific activity was increased at least twofold if the liposomes contained 50 mol% cholesterol. A further increase in specific activity, from 3- to 10-fold, was achieved by fractionation of the membranes on a Renografin gradient before reconstitution. Examination of the fractions from the Renografin gradient by sodium dodecyl sulfate-gel electrophoresis showed a parallel enrichment of glucose transport activity and a number of proteins including one with an apparent Mr of ca. 60,000, which might be the glucose transporter. Finally, preliminary kinetic analysis of glucose transport activity in vesicles reconstituted at a high lipid-to-protein ratio gave a Vmax of ca. 2.8 mumol/mg of protein per min at 23 degrees C and a Km of ca. 8 mM. The latter value corresponds to the kinase-independent, low-affinity component of glucose transport observed in wild-type cells.     

Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin.

We have examined the effects of cyclosporin A (CsA) and a series of CsA analogs that bind with decreasing affinity to cyclophilin, to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4; LTC4) mediators of inflammatory reactions from human basophils. CsA (8 to 800 nM) concentration-dependently inhibited (5 to 60%) histamine release from peripheral blood basophils challenged with anti-IgE. CsA was more potent (92.6 +/- 1.8 vs 59.1 +/- 4.5%; p less than 0.001) and, at low concentrations, more effective when the channel-operated influx of Ca2+ was bypassed by the ionophore A23187 (IC40 = 24.1 +/- 3.9 vs 105.5 +/- 22.2 nM; p less than 0.05). CsA had no effect on the release of histamine caused by phorbol myristate and bryostatin 1 that activate different isoforms of protein kinase C. Inhibition of histamine release from basophils challenged with anti-IgE was not abolished by washing (three times) the cells before anti-IgE challenge. CsA also inhibited the de novo synthesis of LTC4 from basophils challenged with anti-IgE. The inhibitory effect of CsA was very rapid, and the drug, added from 1 to 10 min during the reaction, inhibited the ongoing release of histamine caused by anti-IgE and by A23187. The experiments with CsA analogs (CsG, CsC, CsD, and CsH) showed that CsH, which has an extremely low affinity for cyclophilin, has no effect on basophil mediator release. In addition, there is a significant correlation between the concentrations of CsA, G, C, and D that inhibited by 30% the histamine release induced by anti-IgE (r = 0.99; p less than 0.001) and by A23187 (r = 0.87; p less than 0.001) and their affinity for cyclophilin.     

Purification to homogeneity and biochemical characterization of two suppressor factors from human malignant T-cells

A cell clone (GI-CO-T-9) derived from a long term T-cell culture (PF-382), established from a patient affected by acute T-lymphoblastic leukemia (T-ALL), was selected for the presence in the culture medium of factors suppressing T-cell proliferation. The crude supernatant has been subjected to a multi-step chromatographic fractioning, including: preparative gel permeation, anion exchange, and hydrophobic interaction High Performance Liquid Chromatography (HPLC). The highly purified material was characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE), revealing single bands of 115 Kd and 80 Kd. The isoelectric points (pI), determined by flat-bed isoelectric-focusing, were 7.4 for High Molecular Weight Suppressor Factor (HMWSF) and 3.5-3.6 for Low Molecular Weight Suppressor Factor (LMWSF).     

The <Emphasis Type="Italic">Mycobacterium marinum mel2</Emphasis> locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species

Background  

Mycobacteria have developed a number of pathways that provide partial protection against both reactive oxygen species (ROS) and reactive nitrogen species (RNS). We recently identified a locus in Mycobacterium marinum, mel2, that plays a role during infection of macrophages. The molecular mechanism of mel2 action is not well understood.