Regulation of transferrin receptor cycling by protein kinase C is independent of receptor phosphorylation at serine 24 in Swiss 3T3 fibroblasts

Treatment of Swiss 3T3 fibroblasts with tumor-promoting phorbol diester or with platelet-derived growth factor caused the phosphorylation of the transferrin receptor by protein kinase C (Ca2+/phospholipid-dependent enzyme) at serine 24 and increased the cell surface expression of the transferrin receptor. The hypothesis that the regulation of transferrin receptor cycling by protein kinase C is causally related to the phosphorylation of the receptor at serine 24 was critically tested. Site-directed mutagenesis of the human transferrin receptor cDNA was used to substitute serine 24 with threonine or alanine residues in order to create phosphorylation defective receptors. Wild-type and mutated transferrin receptors were expressed in Swiss 3T3 fibroblasts using the retrovirus vector pZipNeoSV (X). These receptors were functionally active and caused the receptor-mediated endocytosis of diferric transferrin. Incubation of the fibroblasts with phorbol diester caused the phosphorylation of the wild-type (Ser-24) human transferrin receptor, but this treatment did not result in the phosphorylation of the mutated (Ala-24 and Thr-24) receptors. The cycling of the phosphorylation defective receptors was regulated by phorbol diester and platelet-derived growth factor in a manner similar to that observed for the wild-type receptor. We conclude that the regulation of transferrin receptor cycling by protein kinase C is independent of receptor phosphorylation at serine 24 in Swiss 3T3 fibroblasts.     

Retinol and extracellular collagen matrices modulate hepatic Ito cell collagen phenotype and cellular retinol binding protein levels

The hepatic vitamin A-storing Ito cell has been implicated as a causative cell in hepatic fibrogenesis. Using a modification of a recent method (Friedman, S. L., Roll, F. J., Boyles, J., and Bissell, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8681-8685), rat Ito cells were isolated and passaged in vitro on collagen-coated plastic dishes through cell generation 40-50. The collagen synthetic phenotype for Ito cells grown on various extracellular matrices was demonstrated by immunofluorescence and quantitated by competition enzyme-linked immunosorbent assays. When grown on a type I collagen matrix, Ito cells produced type IV greater than type III greater than type I collagen. When grown on a type IV collagen matrix, the cells produced relatively equal amounts of types I and III collagen. The absolute amounts of type I collagen produced were greater when cells were grown on type IV versus type I matrix. When 10(-5) M retinol was added to cell cultures, there was a uniform increase in type III collagen regardless of matrix type but a decrease in type I collagen when cells were grown on a type IV matrix and a large increase in type I collagen when cells were grown on a type I collagen matrix. The levels of cellular retinol binding protein, a key cytosolic retinol transport protein, were quantitated by high performance liquid chromatography and compared for cells grown on type I versus type IV collagen matrices. It was found that cells on a type I matrix contain 4.96 +/- 2.8 times more cellular retinol binding protein than do cells grown on a type IV matrix. In conclusion, Ito cell collagen synthesis may be altered by underlying extracellular matrix and exogenous retinol. This in vitro culture system should allow the study of regulatory factors and possible therapeutic anti-fibrogenic mediators.     

Ornithine decarboxylase from Neurospora crassa. Purification, characterization, and regulation by inactivation

Ornithine decarboxylase, a highly regulated enzyme of the polyamine pathway, was purified 670-fold from mycelia of Neurospora crassa that were highly augmented for enzyme activity. The enzyme is significantly different from those reported from three other lower eucaryotic organisms: Saccharomyces cerevisiae, Physarum polycephalum, and Tetrahymena pyriformis. Instead, the enzyme closely resembles the enzymes from mammals. The Mr = 110,000 enzyme is a dimer of 53,000 Da subunits, with a specific activity of 2,610 mumol per h per mg of protein. Antisera were raised to the purified enzyme and were rendered highly specific by cross-absorption with extracts of a mutant strain lacking ornithine decarboxylase protein. With the antisera, we show that the inactivation of the enzyme in response to polyamines is proportional to the loss of ornithine decarboxylase protein over almost 2 orders of magnitude. This is similar to the inactivation process in certain mammalian tissues, and different from the process in S. cerevisiae and P. polycephalum, in which enzyme modification, without proportional loss of antigen, accompanies enzyme inactivation. The N. crassa enzyme is therefore suitable as a microbial model for studies of the molecular regulation of the mammalian enzyme.     

Regulation of Dipeptidyl Aminopeptidase I and Angiotensin Converting Enzyme Activities in Cultured Murine Brain Cells by Cortisol and Thyroid Hormone

Cultures of dissociated brain cells from 15-day-old fetal mice were grown in the presence and absence of 20 or 50 nM triiodothyronine (T3), 30 or 300 nM cortisol, and 30 nM cortisol plus 50 nM T3 added to chemically defined media or in media supplemented with 15% serum from control and hypothyroid calves. The specific activities of five lysosomal enzymes--N-acetyl galactosaminidase, beta-glucuronidase, beta-galactosidase, cathepsin B, and dipeptidyl aminopeptidase I (DAP-I)--were higher in cells grown in calf serum than in cells grown in defined media. Of these enzymes, only DAP-I was elevated in activity when the cells were grown in hypothyroid calf serum instead of control calf serum. Elevation of DAP-I activity was reversed by addition of 20 nM T3 to hypothyroid calf serum. The enzymatic properties of DAP-I were similar whether the cells were grown in control or hypothyroid calf serum and were similar to those reported for human fibroblasts and the purified enzyme. When the cells were grown in defined media, cortisol decreased the activities of all lysosomal enzymes, with 300 nM cortisol being more effective than 30 nM cortisol. Addition of 50 nM T3 to 30 nM cortisol decreased DAP-I activity more than 30 nM cortisol alone, but 50 nM T3 alone in defined media did not alter DAP-I levels. The reduction of DAP-I activity in these cells by T3 required cortisol, unidentified components in serum, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Fibronectin is stored but not synthesized in mature human peripheral blood granulocytes

To investigate whether peripheral blood granulocytes can synthesize the adhesive glycoprotein, fibronectin, we sought to demonstrate the presence of messenger RNA coding for fibronectin within mature circulating granulocytes. Polyadenylated-enriched RNA was isolated from human peripheral blood granulocytes, human skin fibroblasts (synthesize fibronectin) and HeLa cells (lack fibronectin) and probed with a cDNA clone coding for the cell attachment domain of fibronectin. Hybridization of a fibronectin cDNA fragment occurred with fibroblast RNA but did not occur with granulocyte RNA despite a 100 fold excess granulocyte RNA. Incubation of granulocytes with n-formyl methionyl leucyl phenylalanine, a chemotactic peptide known to augment the release of fibronectin from granulocytes, failed to induce detectable levels of mRNA for fibronectin in granulocytes. There was no difference in the quantity of fibronectin released from chemotactic peptide-stimulated granulocytes pre-incubated in the presence or absence of the protein synthesis inhibitor, cycloheximide, suggesting that fibronectin exists in a stored form in granulocytes. These data suggest that fibronectin in mature granulocytes is the product of synthesis during early myeloid maturation.     

Stabilization of Z-RNA by chemical bromination and its recognition by anti-Z-DNA antibodies

Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. More extensive bromination of poly[r(C-G)] (greater than 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37 degrees C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', and CH1' resonances in both the A- and Z-forms. GH8----GH1' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 degrees C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., & Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York].(ABSTRACT TRUNCATED AT 250 WORDS)     

Current utilization and further development of the palmyra palm (Borassus flabellifer L., Arecaceae) in Tamil Nadu state,India

The palmyra palm Borassus flabellifer,), a multipurpose tree of great utility, occurs extensively in Tamil Nadu state, India. Figuring in history, literature, and folklore of the state, it is exploited for food from the fruit and tuberous seedlings; beverage and sugar from the sap; fiber from the leaf and leafbase for brushes, cordage, weaving, and plaiting; trunk wood for construction and fuel; and numerous minor products. Increasing exploitation of the palmyra threatens the future supply of palm raw materials so important to rural populations. Integrated development of palmyra products for local and export markets, as well as management/conservation measures, are needed both to maximize the economic value of the products and to assure sustained yield from native stands.     

Apolipoprotein composition of bovine lipoproteins isolated by gel filtration chromatography

1. Bovine lipoproteins were isolated from plasma by gel filtration and apolipoprotein composition determined by SDS-polyacrylamide gel electrophoresis. 2. Bovine triglyceride-rich lipoproteins contained a novel low mol. wt protein Mr = 22,000 and low mol. wt proteins that may be analogous to non-ruminant apolipoproteins A-I, A-IV, and E. 3. Apolipoprotein C appeared to be a minor constituent of bovine triglyceride-rich lipoproteins. 4. Triglyceride-rich lipoproteins contained two high mol. wt proteins of approx. Mr = 220,000 and 290,000. 5. The predominant bovine low density lipoprotein apolipoprotein was approx. Mr = 290,000, however, greater then 25 proteins were often observed between Mr = 110,000 and 370,000. 6. Bovine high density lipoprotein contained proteins analogous to apolipoprotein A-I and C apolipoproteins. 7. Differences in apolipoprotein profiles between non-lactating and lactating cows were not apparent.     

Isolation ofAcholeplasma oculi from human amniotic fluid in early pregnancy

Acholeplasmas have been isolated from a variety of animals, insects, and plants, but onlyAcholeplasma laidlawii has previously been found in humans. We have isolatedAcholeplasma oculi in pure culture from the amniotic fluid of a woman at 19 weeks of gestation. The organism was positively identified by growth inhibition, epi-immunofluorescence, and arbutin hydrolysis. Demonstration of organisms directly in amniotic fluid by DNA fluorochrome and immunofluorescence staining provided additional evidence that the isolate was genuine and not a medium contaminant. The remainder of the pregnancy was unremarkable, and a full-term male infant was delivered without complications. Even though there is some evidence possibly associatingA. oculi with various diseases in livestock, the prevalence and significance ofA. oculi in humans has not been determined.