The precursor for transforming growth factor-alpha (TGF-alpha) is a membrane glycoprotein that can establish contact with epidermal growth factor/TGF-alpha receptors on adjacent cells or can be cleaved to release TGF-alpha that diffuses into the medium. Cleavage of pro-TGF-alpha occurs at Ala/Leu-Ala/Leu-Ala-Val-Val sites located at each end of the mature TGF-alpha sequence. To characterize the cleavage process of pro-TGF-alpha and the role of glycosylation in this process, we have introduced a pro-TGF-alpha expression vector in wild type Chinese hamster ovary (CHO) cells and in the mutant CHO cell clone ldlD that has a reversible defect in protein glycosylation. Analysis of metabolically labeled and cell surface-labeled products immunoprecipitated with antibodies directed against the extracellular TGF-alpha sequence and the cytoplasmic pro-TGF-alpha C-terminal domain shows that cleavage of pro-TGF-alpha in wild type CHO cells occurs in two steps. Both processing steps occur after pro-TGF-alpha reaches the cell surface. In the first step, pro-TGF-alpha rapidly (t1/2 = 30 min) loses the amino-terminal segment that precedes the TGF-alpha sequence. In the second step, pro-TGF-alpha is cleaved at the carboxyl terminus of the TGF-alpha sequence releasing this factor into the medium. This second step is slow (t1/2 = 2 h). The action of pancreatic elastase added to CHO-TGF-alpha cells mimics the first step but not the second one. Synthesis, cell surface exposure, rate of cleavage, and generation of bioactive TGF-alpha in ldlD-TGF-alpha cells are not markedly affected by the lack of N-acetylgalactosamine-dependent protein O-glycosylation or galactose-dependent glycan chain modification. The results indicate that, despite their similarity in amino acid sequence, the two cleavage sites that flank TGF-alpha may be processed with different kinetics which can lead to retention of pro-TGF-alpha on the cell surface.

Amino Acid Sequence↗

PubMed1988

Rhabdomyolysis, oedema and arterial hypertension: different syndromes related to topical use of 9-alpha-fluoroprednisolone.

Over a one year period we detected five cases of iatrogenic mineralocorticoidism secondary to topical application of creams containing 9-alpha-fluoro-prednisolone. Although the same product was involved in all cases, the clinical features differed and included two cases of myopathy and hypokalemic rhabdomyolysis, one of oedema and two of arterial hypertension. Discontinuation of treatment and administration of potassium supplements produced a rapid recovery and all patients remain well six months later.

Adolescent↗

PubMed1987

Ribosomal proteins EL11 from Escherichia coli and L15 from Saccharomyces cerevisiae bind to the same site in both yeast 26 S and mouse 28 S rRNA.

The heterologous interaction of Escherichia coli ribosomal protein EL11 with yeast 26 S and mouse 28 S rRNA was studied by analysing the ability of this protein to form a specific complex with various synthetic rRNA fragments that span the structural equivalent of the EL11 binding site present in these eukaryotic rRNAs. The fragments were obtained by SP6 polymerase-directed in-vitro run-off transcription of parts of the yeast or mouse large rRNA gene cloned behind the SP6 promoter. EL11 was found to protect an oligonucleotide fragment of 63 nucleotides from both the yeast and mouse transcripts against digestion by RNase T1. In both cases, the position of this fragment in the L-rRNA sequence coincides almost exactly with that of the fragment previously found to be protected by EL11 in E. coli 23 S rRNA. Moreover, the protected yeast fragment was shown to be able to re-bind to EL11 by a nitrocellulose filter binding assay. A ribosomal protein preparation from Saccharomyces cerevisiae containing L15 (YL23) as well as the acidic proteins L44', L44 and L45 protects exactly the same oligonucleotide fragment as does EL11 in both the yeast and mouse transcripts. Evidence is provided that L15, which is known to be structurally and functionally equivalent to EL11, is the rRNA-binding protein in this preparation. Thus the structural equivalent of the EL11 binding site present in yeast 26 S rRNA constitutes the second example of functional conservation of a ribosomal protein-binding site on rRNA between prokaryotes and eukaryotes.

Animals↗

PubMedDate not supplied

Ruminations regarding the design of small mixtures for biological testing.

Synthesis and screening of compound mixtures offer avenues to increase throughput and reduce cycle time in the discovery of new drugs and agrochemicals. Equations are derived which show that the efficiency of synthesis and screening of mixtures is a function of the screening hit rate and the number of compounds in each mixture when simple one-step deconvolution by retesting the individual compounds in each active mixture is employed. Values of hit rate and number of compounds in each mixture which afford various levels of increased efficiency are delineated. Two-step deconvolution, in which the active mixtures from the first round of testing are subdivided into mixtures with fewer compounds for a second round of mixture screening prior to final testing of individual compounds, is shown to be more efficient than simple one-step deconvolution under most conditions. For optimum efficiency, the number of compounds in each mixture in the second round testing should be the square root of the number of compounds in each mixture in the first round. At high hit rates the efficiency of the double scan or indexed approach to deconvolution is shown to be higher than that of simple deconvolution. This discussion is oriented mainly toward mixtures of 4-20 compounds and screens which give hit rates in the 1-10% range. The equations describing efficiency are applied in the context of a 49-member amide library produced as mixtures of seven compounds. This library includes the commercial herbicide pronamide and was screened for herbicidal and insecticidal utility.

Combinatorial Chemistry Techniques↗