Base pairing in wheat germ ribosomal 5S RNA as measured by ultraviolet absorption, circular dichroism, and Fourier-transform infrared spectrometry

Ultraviolet absorption (UV) and circular dichroism (CD) spectra of wheat germ 5S RNA, when compared to tRNAPhe, indicate a largely base-paired and base-stacked helical structure, containing up to 36 base pairs. Fourier-transform infrared (FT-IR) spectra of tRNAPhe and wheat germ ribosomal 5S RNA have been acquired at 30 and 90 degrees C. From the difference of the FT-IR spectra between 90 and 30 degrees C, the number of base pairs in both RNAs was determined by modification of a previously published procedure [Burkey, K. O., Marshall, A. G., & Alben, J. O. (1983) Biochemistry 22, 4223-4229]. The base-pair composition and total base-pair number from FT-IR data are now consistent for the first time with optical (UV, CD, Raman) and NMR results for ribosomal 5S RNA. Without added Mg2+, tRNAPhe gave 18 +/- 2 base pairs [7 A-U and 11 G-C], in good agreement with the number of secondary base pairs from X-ray crystallography [8 A-U, 12 G-C, and 1 G-U]. Within the 10% precision of the FT-IR method, wheat germ 5S RNA exhibits essentially the same number of base pairs [14 A-U, 17 G-C, and 5 G-U; for a total of 36] in the absence of Mg2+ as in the presence of Mg2+ [14 A-U, 18 G-C, and 3 G-U; for a total of 35], in agreement with the UV hyperchromism estimate of G-C/(A-U + G-C) = 0.58.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stable isotope ratio as a tracer of mangrove carbon in Malaysian ecosystems

Summary The ratio of stable carbon isotopes (¹³C) in plants and animals from Malaysian mangrove swamps, coastal inlets, and offshore waters was determined. Vascular plants of the swamps were isotopically distinct ( x±s.d.=-27.1±1.2) from plankton (-21.0±0.3) and other algae (-18.7±2.2). Animals from the swamps (-20.9±4.1) and inlets (-19.8±2.5) had a wide range of isotope ratios (-28.6 to-15.4), indicating consumption of both mangrove and algal carbon. Several commercially important species of bivalves, shrimp, crabs, and fish obtained carbon from mangrove trees. Mangrove carbon was carried offshore as detritus and was isotopically distinguishable in suspended particulate matter and sediments. Animals collected from 2 to 18 km offshore, however, showed no isotopic evidence of mangrove carbon assimilation, with ratios (-16.5±1.1, range-19.1 to-13.1) virtually identical to those reported for similar animals from other plankton-based ecosystems. Within groups of animals, isotope ratios reflected intergencric and interspecific differences in feeding and trophic position. In particular, there was a trend to less negative ratios with increasing trophic level.     

Characterization and partial purification of keratinocyte growth factor from the hypothalamus

An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.     

Exclusion of erythrocyte-specific membrane proteins from clathrin- coated pits during differentiation of human erythroleukemic cells

When human erythroleukemic cells are induced to differentiate, they produce globin and redistribute glycophorin and spectrin to one pole of the cell. This process was accompanied by an alteration in the clathrin-coated pits at the cell surface. In nondifferentiating cells, receptors for Concanavalin A have been shown, using electron microscopy, to be concentrated into coated pits and rapidly internalized. Glycophorin was also internalized via coated pits, but was not greatly concentrated into these portions of the surface membrane. Ligands attached to glycophorin were, therefore, cleared from the cell surface more slowly than Concanavalin A. In nondifferentiating cells, immunoelectron microscopy showed that spectrin is largely excluded from coated pits. After erythroid differentiation proceeded for several days, glycophorin was totally excluded from the coated pits along with spectrin. This did not reflect a general cessation of endocytosis, however, because Concanavalin A receptors continued to be internalized. It is possible that the specific exclusion of glycophorin from coated pits is part of the remodeling process that occurs when the precursor cell membrane differentiates into that of the mature erythrocyte.     

High-speed preparative-scale separation and purification of ribosomal 5S and 5.8S RNA's via Sephacryl S-300 gel filtration chromatography

In this work we present a rapid and economical alternative to Sephadex and high performance size exclusion chromatography (HPSEC) for preparative-scale separation and purification of low molecular weight RNA's: 5.8S RNA, 5S RNA, and tRNA's. These three RNA species can be well resolved from each other and from higher molecular weight RNA species via Sephacryl S-300 gel filtration chromatography under mild eluting conditions: 10 mM Tris-HCl buffer, pH 7.5, containing 1.0 M NaCl. For a sample load of about 250 mg, the resolving power of a Sephacryl S-300 column (78 X 3.2 cm) is comparable to that of a 4.5 times larger Sephadex G-75 column (144 X 5 cm). Moreover, the total separation period is 2.5 times shorter for the Sephacryl method. Up to 500 mg or more of crude ribosomal RNA mixtures could be separated via two Sephacryl S-300 columns operated in tandem.     

Multiline varieties and disease control

Summary Existing theoretical models have led to conflicting predictions concerning the likely effect of the widespread use of dirty crop multilines on the evolution of virulence in pathogen populations. Here we attempt to clarify these problems by extending existing models to include selection against unnecessary genes for virulence at two different stages in the life cycle of the pathogen. The results of these studies indicate that the stage of the life cycle at which selection occurs can significantly influence the evolution of virulence in pathogen populations growing on multiline varieties.     

Human alpha- and beta-CGRP and rat alpha-CGRP are coronary vasodilators in the rat

The effects of the recently described human alpha-calcitonin gene-related peptide (CGRP), human beta-CGRP and rat alpha-CGRP have been compared with those of the vasodilator sodium nitroprusside, on the rat and rabbit isolated heart. Hearts were perfused at constant flow and [Arg8]-vasopressin was used to increase coronary perfusion pressure. In the rat heart, the order of potency for evoking cumulative dose-dependent falls in perfusion pressure was human beta-CGRP greater than rat alpha-CGRP greater than human alpha-CGRP greater than sodium nitroprusside. In the same preparations the three CGRPs (but not sodium nitroprusside) elicited cumulative dose-related increases in heart rate. In the rabbit heart the order of potency for vasodilatation was rat alpha-CGRP greater than human alpha-CGRP greater than sodium nitroprusside. In marked contrast to results from the rat, neither rat alpha-CGRP nor human alpha-CGRP altered heart rate in the rabbit isolated heart. These results show that human alpha- and beta-CGRP and rat alpha-CGRP are vasodilators in the coronary vasculature, but that there is species variation as CGRP had a positive chronotropic effect in the rat heart but not in the rabbit heart.     

Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor.

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.     

The effect of papain upon proline and sodium transport of rat renal brush-border membrane vesicles

Treatment of renal brush-border membrane vesicles with papain resulted in the removal of the activity of maltase, gamma-glutamyl transpeptidase and leucine aminopeptidase by 85, 50 and 75%, respectively. Stripping of these membrane enzyme activities constituted about 2% of the total membrane proteins and resulted in a widespread diminution in the ability of a variety of amino acids and sugars to be taken up by the membrane vesicles which remained osmotically responsive. Kinetic analysis of the uptake of proline, which was shown previously to be transported by both sodium-dependent and sodium-independent systems, revealed that the Vmax for the sodium-dependent system and Km for the sodium-independent system were halved, but other parameters were not affected indicating that the papain treatment altered sodium-gradient-stimulated entry and the affinity of the sodium-gradient-independent system for proline. Experiments on sodium entry and efflux demonstrate a marked enhancement of flux, so that equilibration of the sodium gradient occurred about 5-times more rapidly than in untreated vesicles. This occurred without any change in the osmotic properties of the vesicle with regard to sodium or amino acid uptake. Studies of fluorescence polarization suggest that incubation with papain does not alter the lipid domains of the membrane.