Direct observation of orbital ordering in La0.5Sr1.5MnO4 using soft x-ray diffraction.

We report the first direct resonant soft x-ray scattering observations of orbital ordering. We have studied the low temperature phase of La0.5Sr1.5MnO4, a compound that displays charge and orbital ordering. Previous claims of orbital ordering in such materials have relied on observations at the manganese K edge. These claims have been questioned in several theoretical studies. Instead we have employed resonant soft x-ray scattering at the manganese L(III) and L(II) edges which probes the orbital ordering directly. Energy scans at constant wave vector are compared to theoretical predictions and suggest that at all temperatures there are two separate contributions to the scattering: direct orbital ordering and strong cooperative Jahn-Teller distortions of the Mn3+ ions.

Soft X-ray resonant magnetic diffraction.

We have conducted the first soft x-ray diffraction experiments from a bulk single crystal, studying the bilayer manganite La2-2xSr1+2xMn2O7 with x=0.475 in which we were able to access the (002) Bragg reflection using soft x rays. The Bragg reflection displays a strong resonant enhancement at the L(III) and L(II) manganese absorption edges. We demonstrate that the resonant enhancement of the magnetic diffraction of the (001) is extremely large, indeed so large that it exceeds that of the nonresonant Bragg diffraction. Resonant soft x-ray scattering of 3d transition metal oxides is the only technique for the atomic selective measurement of spin, charge, and orbital correlations in materials, such as high temperature superconductors, colossal magnetoresistance manganites, and charge stripe nickelates.

Purified herpes simplex thymidine kinase Retrovector particles. I. In vitro characterization, in situ transduction efficiency, and histopathological analyses of gene therapy-treated brain tumors.

Replication-defective, highly purified retroviral vectors (Retrovector), at titers of 10(8) colony forming units/mL, were prepared that conferred either beta-galactosidase or herpes simplex thymidine kinase (HSV-TK) activity. 9L gliosarcoma cells, transduced efficiently in vitro, were highly sensitive to ganciclovir (GCV). The mean frequency of in situ transduction, measured by flow cytometry of single-cell tumor suspensions isolated from rat brains, was 3.2 +/- 0.6%; similar assessments were made by staining of beta-galactosidase or by immunohistochemistry with anti-HSV-TK. In vitro HSV-TK-transduced and G418-selected 9L-TK gliosarcoma tumors treated with GCV were eradicated in approximately 53% of the animals (10/19) at day 26, however, 89% (17/19) histologically showed < 1% tumor volume. Histologic evaluation at day 26 of animals with established 9L tumors treated with intralesional injection of HSV-TK vector followed by GCV treatment showed that 29% (4/14) had no tumor; 50% (7/14) had < 1% tumor volume. Regression of tumors proceeded over the time since the complete rate was increased at day 60. Neither HSV-TK vector particles nor GCV alone altered the histological profile of 9L tumors, but substantial numbers of CD4+ and CD8+ lymphocytes infiltrated the tumors of animals treated with both. In cured animals, the former tumor bed contained cell debris, immune cells, and fibroblasts and was without damage to adjacent brain. The efficacy of suicide gene therapy for rat gliosarcoma using highly purified virion vectors approaches that of packaging cell lines.

Comparison of the 5' end of the rat and mouse cystathionine beta-synthase genes.

We have isolated and characterized a genomic fragment encompassing the first six exons and 2.6 kbp 5' flanking sequence of the rat cystathionine beta-synthase (CBS) gene. A previously unknown exon approximately 3 kbp upstream of exon 1 was identified. The transcription start site was mapped to approximately 3 kbp upstream from the translation start codon and contains a consensus cap signal. The putative promoter region contains three GC boxes, in both orientations, and no TATA box. We have also compared a 1171-bp-long DNA sequence of the 5' end of the rat CBS gene with the homologous mouse region of 1125 bp. We found two homologous Sp1 sites in the mouse gene and an overall sequence conservation of 70% with 88-89% similarity in the 80-bp regions surrounding the intron 0 splice sites.

Human cystathionine beta-synthase cDNA: sequence, alternative splicing and expression in cultured cells.

Cystathionine beta-synthase (CBS) deficiency is the major cause of homocystinuria in humans. The most frequent symptoms of homocystinuria include: dislocated optic lenses, vascular disorders, skeletal abnormalities and mental retardation. Patients with this deficiency have elevated levels of homocyst(e)ine, methionine and low cysteine in their body fluids. These abnormal levels often partially or fully normalize upon treatment with pharmacological doses of vitamin B6. To investigate the molecular and biochemical basis for these conditions, it was necessary to determine the nucleotide and polypeptide sequence of CBS. We report here the human CBS cDNA sequence of 2,554 nucleotides encoding the CBS subunit of 551 amino acids. An intron of 214 bp appears to be retained in the 3'-untranslated region of most of the fibroblast and liver mRNA. We also report a frequent Mspl polymorphism in the 3'-untranslated sequence and two synonymous mutations in the coding region: 699C/T (Y233Y) and 1080C/T (A360A). The amino acid sequence similarity of human and rat CBS is greater than 90%; the enzyme also exhibits 52% similarity to O-acetylserine(thiol)-lyase from bacteria and plants. Lastly, we demonstrate that expression of the human enzyme in CHO cells yields enzymatically active protein of the expected size with a half-life of approximately 14 hrs.

Rat cystathionine beta-synthase: expression of four alternatively spliced isoforms in transfected cultured cells.

The gene for rat cystathionine beta-synthase consists of 17 exons. Its transcripts are alternatively spliced, forming four distinct mRNA species. Type III consists of exons 1 through 12, 14, 15, and 17; type I also contains exon 16. The open reading frame of type IV spans exons 1-->13; type II, 3-->13. We cloned the corresponding cDNAs into appropriate expression vectors and inserted the constructs into Escherichia coli (I, III, and IV) and Chinese hamster (CH) cells (I through IV); all sequences were transcribed and translated. Catalytic activity was observed only for types I and III in lysates of transfected CH cells and transformed E. coli. The catalytic and kinetic properties of I and III were identical despite their structural difference (exon 16). Both isoforms exhibited 6 mM Km constants for homocysteine which were reduced approximately eightfold by AdoMet; this elucidates the mechanism by which AdoMet regulates synthase activity. The four isoforms were differentially degraded by transfected cultured cells. Type III (t1/2 = 18 h) was degraded at 1/3 the rate of type I (t1/2 = 6 h); thus the 14 amino acid residues encoded by exon 16 appear to enhance degradation of CBS. The half-lives of both types II and IV were markedly shorter (ca. 1 h). Western blots comparing rat liver to lysates from transfected CH cells revealed that hepatocytes express both isoforms. Type III was predominant, as predicted by its longer half-life and more abundant mRNA. PCR analysis of cDNA from various tissues revealed that type III mRNA was preferred in liver, kidney, and heart; equal amounts of I and III were found in brain.

Rat cystathionine beta-synthase. Gene organization and alternative splicing.

We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene. The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons. These are alternatively spliced, forming four distinct mRNAs (types I through IV). The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively. Exons 13 and 16 are used alternatively and mutually exclusively. Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV. Exon 16 is present only in type I. Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver. Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III. These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells. Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms. Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E. coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33%. Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.

Evidence for acceleration of the rate of elongation of tyrosine aminotransferase nascent chains by dibutyryl cyclic AMP.

Analogs of cyclic AMP elevate the synthesis of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5) in cultured hepatoma cells and rat liver at a post-transcriptional level but have no discernible effect on total soluble protein synthesis. In order to determine whether cyclic AMP exerts its effect on a step before or after initiation of the synthesis of this enzyme, we have analyzed the ribosomal transit times for both the aminotransferase and total soluble protein in hepatoma cells incubated in the presence or absence of N(6),O(2)'-dibutyryl cyclic AMP. The time required for one ribosome to translate one subunit of the "average" soluble protein (transit time) was about 2 min in cells incubated with or without the cyclic AMP analog. In contrast, the transit time for tyrosine aminotransferase was found to be reduced from 5-8 min under basal conditions to as low as 45 sec after exposure to dibutyryl cyclic AMP. Although the degree of effect varied from experiment to experiment, the relative rate of aminotransferase nascent chain elongation was found to be proportional to the stimulation of its activity. In contrast, dexamethasone did not alter the rate of aminotransferase elongation even though it elevated enzyme activity between 5- and 10-fold. These data are consistent with the hypothesis that induction of tyrosine aminotransferase with cyclic AMP analogs occurs by stimulation of the rate at which ribosomes translate pre-existing mRNA in contrast to adrenal steroids which act by increasing the level of translatable mRNA coding for this enzyme.

Glucocorticoid control of the development of tryptophan oxygenase in the young rat.

Tryptophan oxygenase activity follows a characteristic developmental pattern in the young rat, being absent until about 2 weeks of life, then attaining adult levels by the 3rd week. We have investigated the factors which control this process and determined that the increase in enzymatic activity results from increased glucocorticoid release between the 14th and 21st days. This conclusion was based on our observation that adrenalectomy completely arrests the development of the enzymatic activity. Twenty-one-day-old animals which were adrenalectomized on the 10th day, and thus possess no demonstrable enzymatic activity, could respond to cortisol treatment with an elevation of tryptophan oxygenase activity. Administration of either cycloheximide or actinomycin D completely inhibited this response, suggesting that both protein synthesis and RNA synthesis were requisite for the development of the enzyme. Immunochemical studies revealed that the enzyme found in the young animal was identical with that extracted from the adult rat; furthermore, the increase in activity observed in the developing rat is the result of increased enzyme protein levels. Direct measurement of the synthetic and degradative rates showed the accumulation of enzyme protein to depend upon increased synthetic activity, and not upon decreased enzyme degradation.

Effects of 6- and 8-substituted analogs of adenosine 3':5'-monophosphate on phosphoenolpyruvate carboxykinase and tyrosine aminotransferase in hepatoma cell cultures.

A variety of 6- and 8-substituted analogs of cAMP (cyclic adenosine 3:5-monophosphate) have been tested for their ability to increase activity of tyrosine aminotransferase (EC 2.6.1.5) in cultured Reuber H35 hepatoma cells. Some analogs, particularly the 8-thio-substituted ones, produced effects approximately equivalent to those generated by N-6, O2'-dibutyryl cAMP. In contrast, cAMP and its O-2-monobutyryl derivative were relatively ineffective even at very high concentrations, whereas three other analogs actually depressed the activity of the aminotransferase. Changes in enzyme activity generated by the various analogs were paralleled closely by changes in the relative rate of aminotransferase synthesis. An excellent correlation was found to exist between the ability of any given analog to influence the activity of tyrosine aminotransferase and that of phosphoenolpyruvate carboxykinase (EC 4.1.1.32). A similar correlation was found to exist between the ability of various analogs to evelate the activity of these enzymes and to inhibit reversibly the growth of H35 cells. Only one of five inhibitors of cAMP phosphodiesterase activity tested produce any increase in aminotransferase activity when added alone. All of the 6- and 8-substituted analogs tested, including noniducers, stimulated f1 histone phosphorylation in crude rat liver extracts with approximately equal potencies. On the other hand, dibutyryl cAMP was only a weak activator of protein kinase in vitro, even though it is a potent enzyme inducer. A possible resolution of this apparent discrepancy has been provided by preliminary analyses of site-specific f1 histone phosphorylation in whole cells. Only compounds active as aminotransferase inducers are capable of stimulating phosphorylation of the serine-37 residue of endogenous f1 histone (3- to 10-fold).