Expression of cloned aromatic L-amino acid decarboxylase in Xenopus laevis oocytes.

Sense mRNA coding for bovine adrenal medulla aromatic L-amino acid decarboxylase (AADC) was expressed following microinjection into Xenopus laevis oocytes. The expressed enzyme activity was stereoselective for L-5-hydroxytryptophan and L-DOPA and blocked by NSD-1015 an inhibitor of AADC. Heating the expressed enzyme at 55 degrees C resulted in a parallel loss of activity towards both substrates. Our findings are consistent with the prevailing notion that a single enzyme is able to decarboxylate both substrates in vivo.     

Accumulation of free amino acids in growing Xenopus laevis oocytes

The sizes of amino acid pools in growing Xenopus laevis oocytes have been measured. The total free amino acid content per oocyte increases nearly 25-fold during oocyte growth. Together, glutamic acid and aspartic acid account for approximately 59-75% of the total amino acid pool in Xenopus oocytes. On the other hand, methionine and cysteine are the least abundant of the amino acids detected, each accounting for less than 0.7% of the total pool in developing oocytes. It is argued that the acid-extractable amino acid pool represents the precursor pool used in protein synthesis.     

The degradation of ribonucleic acids injected into Xenopus laevis oocytes

Different radioactive RNAs were injected into Xenopus laevis oocytes, and their degradation followed with time. Deproteinized ribosomal RNAs and synthetic polynucleotides, with the exception of polyadenylic acid, were degraded rapidly with apparent first order kinetics and half-lives ranging from 1–6 hr. Transfer RNA, poly(A), and ribosomal RNA injected as whole ribosomal particles were quite stable during the period studied (20 hr). Messenger RNAs from Dictyostelium discoideum and Vesicular Stomatitis Virus, which have poly(A) sequences at their 3′ terminus, presented biphasic degradation kinetics. Approximately 60% of these RNAs was degraded in the first 6 hr, whereas the remaining 30–40% was stable for at least 22 hr. Analysis of the stable material by sucrose gradients showed that it had the same sedimentation pattern as the original material, except that it contained, in addition, free poly(A) sequences sedimenting somewhat smaller than 4S. Puromycin treatment of the cells injected with Dictyostelium mRNAs reduced the percentage of stable RNA to 10%, approximately the poly(A) content of these RNAs. Similar treatment with emetine, which also inhibited cellular protein synthesis, did not affect the stable mRNA fraction.     

Actin in Xenopus oocytes

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.     

Lipoxygenase metabolism of polyunsaturated fatty acids in oocytes of the frog Xenopus laevis

Recently, oocytes or eggs of two marine invertebrates have been found to metabolize arachidonic acid to specific monohydroxy products. These studies have prompted our examination of the oocytes of higher organisms. In the present study, oocytes of an amphibian, Xenopus laevis, were examined for their capacity to biosynthesize hydroxyeicosatetraenoic acids (HETEs) and related hydroxy fatty acids. Two hydroxyeicosanoids were formed during incubations of oocyte homogenates with [14C]arachidonic acid; their structures and stereochemistry were determined by high-pressure liquid chromatography, uv spectroscopy, and gas chromatography-mass spectrometry. The compounds were identified as 15(S)- and 12(S)-hydroxyeicosatetraenoic acids. The synthesis of the two HETEs was not blocked by a cyclooxygenase inhibitor, indomethacin (10 microM), or by prior exposure of the oocyte homogenates to carbon monoxide, an inhibitor of cytochrome P450. Furthermore, 12(S)- and 15(S)-hydroperoxyeicosatetraenoic acids were isolated from brief incubations of gel-filtered ammonium sulfate fraction of frog oocyte homogenates; isolation of the hydroperoxide is further support for the existence of 12(S)- and 15(S)-lipoxygenase activities in the oocytes of X. laevis. Other polyunsaturated acids, including C18.2, C18.3, C20.3, C20.5, and C22.6 were also substrates for the lipoxygenase, and in each case the major product was formed by omega 6 oxygenation.     

Transport of amino acids and nucleosides in metaphase-arrested unfertilized oocytes of Xenopus laevis

Metaphase-arrested, unfertilized shed oocytes of Xenopus laevis obtained after hormonal stimulation of the female are able to take up nucleosides (U, T) and amino acids (Ala, Gly, Glu, Gln, Tyr). For alanine, tyrosine, and glutamic acid the transport is uphill. The transport of the amino acids studied is activated by Na+, whereas the uptake of the nucleosides is independent of the Na+ concentration. Ouabain does not inhibit the uptake of amino acids significantly. The uptake of alanine and thymidine is not measurably affected by the presence of the jelly coat.     

L-amino acid oxidases with specificity for basic L-amino acids in cyanobacteria

The two closely related fresh water cyanobacteria Synechococcus elongatus PCC 6301 and Synechococcus elongatus PCC 7942 have previously been shown to constitutively express a FAD-containing L-amino acid oxidase with high specificity for basic L-amino acids (L-arginine being the best substrate). In this paper we show that such an enzyme is also present in the fresh water cyanobacterium Synechococcus cedrorum PCC 6908. In addition, an improved evaluation of the nucleotide/amino acid sequence of the L-amino acid oxidase of Synechococcus elongatus PCC 6301 (encoded by the aoxA gene) with respect to the FAD-binding site and a translocation pathway signal sequence will be given. Moreover, the genome sequences of 24 cyanobacteria will be evaluated for the occurrence of an aoxA-similar gene. In the evaluated cyanobacteria 15 genes encoding an L-amino acid oxidase-similar protein will be found.     

Gene expression in Xenopus oocytes

1. Gene expression in Xenopus oocytes is now an integral part of many molecular cloning strategies. 2. For some genes, such as those encoding the ion channels, this system has emerged as the only available means to authenticate and examine the biological activities of the cloned DNA. 3. This review discusses some of the current applications of Xenopus oocytes in modern molecular biology.     

Site-specific insertion of gene cassettes into integrons

Site-specific insertion of gene cassettes into the insert region of integrons has been demonstrated. Insertion was only observed if the integron DNA integrase was expressed in the recipient cell and if the cassette DNA was ligated prior to transformation. The essential ligation products were resistant to treatment with exonuclease III, indicating that they were closed circular molecules. Insertion of cassettes into integron fragments containing either no insert (one recombination site), or one gene cassette (two recombination sites), was demonstrated. In the latter case, insertion occurred predominantly at the core site located 5′ to the resident cassette, which corresponds to the only site available when no insert is present in the recipient. When DNA molecules including two gene cassettes were used, insertion of only one of the gene cassettes was generally observed, suggesting that resolution of the circular molecule to generate two independent circular cassettes occurred more rapidly than insertion into the recipient integron.     

L-amino acids regulate parathyroid hormone secretion

Parathyroid hormone (PTH) secretion is acutely regulated by the extracellular Ca(2+)-sensing receptor (CaR). Thus, Ca(2+) ions, and to a lesser extent Mg(2+) ions, have been viewed as the principal physiological regulators of PTH secretion. Herein we show that in physiological concentrations, l-amino acids acutely and reversibly activated the extracellular Ca(2+)-sensing receptor in normal human parathyroid cells and inhibited parathyroid hormone secretion. Individual l-amino acids, especially of the aromatic and aliphatic classes, as well as plasma-like amino acid mixtures, stereoselectively mobilized Ca(2+) ions in normal human parathyroid cells in the presence but not the absence of the CaR agonists, extracellular Ca(2+) (Ca(2+)(o)), or spermine. The order of potency was l-Trp = l-Phe > l-His > l-Ala > l-Glu > l-Arg = l-Leu. CaR-active amino acids also acutely and reversibly suppressed PTH secretion at physiological ionized Ca(2+) concentrations. At a Ca(2+)(o) of 1.1 mm and an amino acid concentration of 1 mm, CaR-active amino acids (l-Phe = l-Trp > l-His = l-Ala), but not CaR-inactive amino acids (l-Leu and l-Arg), stereoselectively suppressed PTH secretion by up to 40%, similar to the effect of raising Ca(2+)(o) to 1.2 mm. A physiologically relevant increase in the -fold concentration of the plasma-like amino acid mixture (from 1x to 2x) also reversibly suppressed PTH secretion in the Ca(2+)(o) concentration range 1.05-1.25 mm. In conclusion, l-amino acids acutely and reversibly activate endogenous CaRs and suppress PTH secretion at physiological concentrations. The results indicate that l-amino acids are physiological regulators of PTH secretion and thus whole body calcium metabolism.     

Hyperpolarization-activated chloride currents in Xenopus oocytes

During hyperpolarizing pulses, defolliculated Xenopus oocytes have time- and voltage-dependent inward chloride currents. The currents vary greatly in amplitude from batch to batch; activate slowly and, in general, do not decay; have a selectivity sequence of I- > NO3- > Br- > Cl- > propionate > acetate; are insensitive to Ca2+ and pH; are blocked by Ba2+ and some chloride channel blockers; and have a gating valence of approximately 1.3 charges. In contrast to hyperpolarization- activated chloride currents induced after expression of phospholemman (Palmer, C. J., B. T. Scott, and L. R. Jones. 1991. Journal of Biological Chemistry. 266:11126; Moorman, J. R., C. J. Palmer, J. E. John, J. E. Durieux, and L. R. Jones. 1992. 267:14551), these endogenous currents are smaller; have a different pharmacologic profile; have a lower threshold for activation and lower voltage- sensitivity of activation; have different activation kinetics; and are insensitive to pH. Nonetheless, the endogenous and expressed current share striking similarities. Recordings of macroscopic oocyte currents may be inadequate to determine whether phospholemman is itself an ion channel and not a channel-modulating molecule.     

Cytoskeleton in Xenopus oocytes and eggs

The Xenopus egg is a huge cell divided into compartments with distinct characteristics. The organization of the cytoskeleton reflects both the size of the egg and its regional differences. We review the information concerning the deployment and function of cytoskeletal elements during the changes in cellular organization accompanying oogenesis, oocyte maturation, and following fertilization.     

Mechanism of chromatin assembly in Xenopus oocytes

We have analyzed the chromatin assembly reaction catalyzed by the Xenopus oocyte extract (S-150). A 50 S complex is formed upon mixing the 17 S pUC DNA and the S-150. Mature histones are not detected in this complex, which contains relaxed DNA and protein, and generates subnucleosomal 7 S particles upon digestion with micrococcal nuclease. The relaxed nucleoprotein is gradually supercoiled into nucleosomal chromatin in the S-150, via a pathway that requires ATP and is blocked by novobiocin, and this process is accompanied by the appearance of mature histones H3 and H4. Isolated complexes also supercoil in vitro, which implies the complex is a kit that contains histone precursors, as well as topoisomerases and other enzymes required for assembly. We discuss the biological implications of these findings.     

Synthesis of carp proinsulin in Xenopus oocytes

Total poly(A)-containing RNA from Brockmann boides of carp (Cyprinus carpio) directs the synthesis of authentic carp proinsulin in Xenopus oocytes. Neither preproinsulin nor further processing of the proinsulin to insulin can be detected in the oocytes.     

Induction of membrane excitability in Xenopus oocytes

Immature oocytes from the African toad Xenopus laevis are not known to be excitable cells, which means that they do not generate an action potential in response to small depolarizations. However, a regenerative response is produced if successive depolarizing currents of large magnitude are applied to the oocyte membrane. This response is characterized by the occurrence of a positive transmembrane potential that can last for several minutes. The opening of voltage-dependent channels, highly selective for sodium ions, underlies the depolarization thus obtained. These channels exhibit unconventional electrophysiological and pharmacological properties, which set them apart from other types of voltage-dependent sodium channels found in excitable tissues. The opening of the oocyte sodium channels is a complex process, which includes an induction phase. During this phase, the channels change from an electrical state of inexcitability into an excitable voltage-dependent state. The induction mechanism is modulated by the temperature of the bathing medium, by the activation of enzymes (namely a phospholipase C and a protein kinase C) and by the release of calcium ions from intracellular phosphatidyl-inositol trisphosphate stores. The results summarized in this review point out the possible role that these sodium channels may play in the physiology of the oocyte.