Three cDNA clones, pHGR122, pHGR11, and pHGR74 containing the coding information for abundant mRNAs were identified from a human ovarian granulosa cell cDNA library. Characterization by nucleotide sequencing revealed that pHGR122 was specific for a collagenase inhibitor and pHGR11 for melanoma-associated antigen ME491. Relative quantification by Northern analysis indicated that collagenase inhibitor mRNA is a major species in granulosa cells. This finding provides evidence for the origin of this protein in follicular fluid as a secretory product of granulosa cells. pHGR11 identified melanoma-associated antigen ME491 as the unexpected product of normal, noncarcinogenic, granulosa cells. pHGR74 has the complete coding information for an unknown protein. Three independent experiments: (i) cell-free translation of pHGR74 RNA; (ii) transcription of suitable restriction fragments followed by cell-free translation; (iii) hydrolysis of the cell-free translation product of pHGR74 RNA by endoproteinase Lys-C, identified one open reading frame coding for an acidic, highly hydrophilic protein of 111 amino acid residues. pHGR74 mRNA is expressed in human testis, prostate, seminal vesicle, and ovarian granulosa cells. A comparative Southern analysis indicates pHGR74 mRNA is species specific and encoded by a single-copy gene.

The use of two primers allowed the specific enzymatic amplification of elongation factor 2 starting with total double-stranded cDNA from human ovarian granulosa cells. The amplified DNA fragment with a length of 1765 bp was restricted and sequenced by the shot gun approach. From the sequences obtained from the amplified fragment and the cDNA insert of pHGRSl [Rapp et al. (1988) Biol. Chem. Hoppe-Seyler369,247-250] respectively, the DNA sequence containing the complete coding as well as the 3'-untranslated region was assembled. Vollständige Sequenz der kodierenden Region des menschlichen Elongationsfaktors 2 (EF-2) mittels enzymatischer Amplifizierung der cDNA aus humanen ovariellen GranulosazellenZusammenfassung: Die Verwendung von zwei Primer-Oligonucleotiden ermöglichte die spezifische enzymatische Amplifizierung von ElongationsFaktor-2-cDNA ausgehend von Gesamt-cDNA aus humanen ovariellen Granulosazellen. Das amplifizierte DNA-Fragment wies eine Länge von 1765 Nucleotiden auf. Es wurde durch Restriktionsenzyme fragmentiert. Die erhaltenen Restriktionsmischungen wurden kloniert und nach der "shotgun"-Methode sequenziert. Von der erhaltenen Sequenz des amplifizierten DNA-Fragments und der Sequenz des cDNA-Inserts des Klons pHGR81 m wurde eine DNA-Sequenz zusammengefügt, welche sowohl die vollständige kodierende als auch die 3 f -untranslatierte Region der cDNA für den humanen Elongationsfaktor 2 umfaßte.

((Abstract))The marine tetramic acids melophlin P, Q and R were synthesized for the first time in only four steps. Together with the congenerous melophlins A-C, and G they were also tested for antimicrobial and cytotoxic effects. Melophlins B, C, P, Q and R which share a 5-methyl residue showed some antibacterial activity, mainly in Grampositive bacteria. Melophlins B, C and R which have methyl branched 3-acyl side chains in common, inhibited the growth of cells of human KB-3-1 cervix carcinoma, A-498 kidney carcinoma and U-937 leukemia at IC 50 < 10 µM. They were similar in activity to cisplatin. Melophlin Q, also methyl branched, was astoundingly specific in inhibiting A-498 kidney cancer cells while melophlin P inhibited U-937 leukemia cells particularly well. The position of the methyl branch is decisive for the magnitude of the antiproliferative effect of the melophlin couples B/C and R/Q. 3 IntroductionThe melophlins 1 are N-methyl-3-acyltetramic acids that differ only in the substituents at C(5) (H or Me) of the pyrrolidine-2,4-dione core and in the chain length (C 12 to C 16 ) and branching of the 3-acyl residue (Fig. 1) Results and Discussion ChemistryThe melophlins P (1p), Q (1q) and R (1r) were prepared in four steps as previously described for the congeners B and C (Scheme 1) [11]. N-methylalanine t-butyl ester 2 was reacted with 3, the cumulated ylide Ph 3 P=C=C=O, immobilized on polystyrene [12] to give the tetramate 4 in 92% yield as product of a domino addition / intra-Wittig alkenation process. Since the natural products 1p-r had been shown by oxidative degradation to be 1:1 mixtures of (5R)-and (5S)-stereoisomers [3], racemic 2 was employed. Immobilization of the phosphorus ylide greatly facilitated removal of by-product phosphine oxide. Quantitative cleavage of the ester 4 with trifluoroacetic acid (TFA) gave 1,5-dimethylpyrrolidine-2,4-dione 5. This was 3-acylated under Jones' conditions [13] with BF 3 -diethyl etherate and the respective acyl chloride 6, which was racemic in the case of 6r, to furnish the corresponding 5 BF 2 -adducts 7 in a moderate 20-40% yield under classical thermal but in 30-65% yield under microwave irradiation conditions. The BF 2 -chelates 7 were finally converted to melophlins P, Q or R, respectively, by boiling in methanol. The required chlorides 6q and 6r were prepared from 13-methyltetradecanoic acid (13-MTA) [14] and 12-methyltetradecanoic acid (12-MTA), respectively. To assess the influence of metal chelation on the bioactivity we also prepared a stable Ca(II) complex of Biological evaluationKinases and phosphatases play a pivotal role in the signal transduction of cancer cells. As 3-acyltetramates were frequently reported to inhibit these enzymes, we looked for antiproliferative effects of a representative, structurally diverse subset of melophlins (A-C, G, P-R) in human KB-3-1 cervix, human epithelial A-498 kidney carcinoma, and U-937 leukemia cells as well as L929 mouse fibroblasts (Table 1). In terms of stereochemistry, the compounds were prepared and tes...

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A cDNA clone, pHGRSl, encoding 358 amino-acid residues of the C-terminal region of human elongation factor 2 (EF-2), was isolated from a human ovarian granulosa cell cDNA library. The deduced amino-acid sequence of pHGRSl, when compared with the known identical amino-acid sequences of hamster as well as rat EF-2 revealed a substitution of a glutamine by an alanine residue in the partially determined human sequence. The 15 amino-acid-residue sequence comprising the histidine-715, supposed to be of importance for the biological function of EF-2, is preserved in human EF-2. The coding region of the cDNA insert of pHGRSl displays a homology of 87% to hamster and of 88% to rat EF-2 cDNA. In Northern-transfer analysis, pHGRSl specifically hybridizes with an mRNA species of 3.1 kb. Klonierung und Sequenzanalyse einer cDNA aus humanen ovariellen Granulosa-Zellen mit der kodierenden Information für den C-terminalen Teil des Elongationsfaktors 2Zusammenfassung: Ein cDNA-Klon, pHGRSl, welcher für 358 Aminosäurereste der C-terminalen Region des humanen Elongationsfaktors 2 (EF-2) kodiert, wurde aus einer cDNA-Bank von humanen ovariellen Granulosazellen isoliert. Ein Vergleich der von der Nucleotidsequenz des Klons pHGRSl abgeleiteten Aminosäure-sequenz mit den bekannten, untereinander identischen Aminosäuresequenzen von EF-2 aus Hamster und Ratte, ergab eine Substitution eines Glutamin-durch einen Alaninrest in der humanen Sequenz. Der Bereich von 15 Aminosäureresten, der das Histidin-715 enthält und der für die biologische Funktion besondere Bedeutung hat, ist in der humanen Sequenz konserviert. Die kodierende Region des cDNAInserts von pHGRSl weist eine Homologie von 87% zur Hamster-und von 88% zur Ratten EF-2-Sequenz auf. In der Northern-Analyse hybridisiert pHGRSl spezifisch mit einer mRNASpezies von 3.1 kb.