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L D Smith

Publications and source records attributed to L D Smith.

At least 91 records · Page 5Linked to original sources

Discrete sub-aortic stenosis and ventricular septal defect.

We present details of 15 children, aged 3 months to 11 years, with discrete sub-aortic stenosis and ventricular septal defect. We emphasise a high index of clinical suspicion and echocardiography as the best means of diagnosing this dangerous combination. Physical signs were those of ventricular septal defect in all patients, with auscultatory evidence of additional sub-aortic stenosis in only one. Five patients had a short early diastolic murmur of mild aortic incompetence. The electrocardiograph showed isolated left ventricular hypertrophy in eight patients. Cardiac catheterisation and angiography identified the ventricular septal defect in all cases but detected the sub-aortic stenosis in only eight. Cross sectional echocardiography showed both lesions in all 11 patients to whom it was available.

Aortic Stenosis, Subvalvular↗

Intracellular pH and ribosomal protein S6 phosphorylation: role in translational control in Xenopus oocytes.

The induction of amphibian oocyte maturation with progesterone as well as the activation of sea urchin eggs at the time of fertilization result in increased protein synthesis. The increase in both cases involves the recruitment of maternal mRNA onto polysomes. Further, it has been reported that sea urchin eggs, like full-grown Xenopus oocytes, contain no spare translational capacity based on the observation that injected heterologous mRNA is translated only at the expense of endogenous messages. The nature of the limiting component defined by such experiments is not known, but two factors which have been proposed to play a role in regulating protein synthesis are ribosomal protein S6 phosphorylation and intracellular pH. In the current paper, we review the literature and present new evidence on the roles intracellular pH and S6 phosphorylation have in regulating protein synthesis in Xenopus oocytes. We report that pHi does not increase between stage 3 and stage 6, yet the protein synthetic rate increases at least eight fold during the same period. Hence, we conclude that increasing pHi is not a prerequisite for increasing protein synthesis. Moreover, we present three arguments against increased ribosomal protein S6 phosphorylation being sufficient or necessary for increased protein synthesis S6 phosphorylation being sufficient or necessary for increased protein synthesis in Xenopus oocytes. First, the level of S6 phosphorylation does not increase between stages 4 and 6, a period exhibiting a two to three fold increase in protein synthesis. Second, the injection of globin mRNA into stage-4 oocytes increases total protein synthesis two to three fold, but has no effect on S6 phosphorylation. Third, when the injection of globin mRNA into stage-4 oocytes is followed by an injection of MPF, a dramatic increase in S6 phosphorylation is seen, but total protein synthesis is not further stimulated.

Adenosine Triphosphate↗

Interspersed poly(A) RNAs of amphibian oocytes are not translatable.

The poly(A) RNA of the Xenopus oocytes has been shown to include both single copy and interspersed transcripts. Interspersed maternal poly(A) RNAs contain repetitive sequence elements distributed within regions transcribed from single copy sequences. When renatured these RNAs form partially double-stranded RNA networks, and as shown earlier this can be utilized for preparative separation of interspersed maternal transcripts from maternal transcripts that remain single-stranded after renaturation (Anderson et al., 1982). The translational activity of these RNA fractions was tested in vitro, in wheat germ and reticulocyte systems. While the single-stranded fractions supported protein synthesis, the interspersed oocyte RNAs displayed little translational activity. Translational activity was measured in vivo by injection into the Xenopus oocyte. Oocytes previously injected with globin mRNA were injected with increasing amounts of single-stranded, double-stranded, or denatured double-stranded RNA fractions, and the amount of globin synthesis was determined. It was found that single-stranded RNA competes with globin mRNA for the limited translational apparatus of the oocyte, as manifested by a quantitative reduction of globin synthesis. However, globin synthesis was not affected when double-stranded RNA, either in renatured or denatured form, was injected. We conclude that the interspersed RNAs are not translated within the oocyte. The amount of single and double-stranded RNAs loaded onto polysomes in the injected oocytes was also determined. Sixty seven per cent of radio-iodinated single-stranded RNA pelleted with polysomes in injected oocytes, whereas less than 20% of similarly labeled double-stranded RNA pelleted with polysomes. This value is similar to that obtained when partially hydrolyzed RNA is injected, suggesting again that essentially none of the interspersed RNA is translated in vivo. The significance of these findings in relation to translational regulation during oogenesis and early development is discussed.

Animals↗

SAMP lyase and AMP deaminase activity in rat parenchymal and kupffer cells in hepatocarcinogenesis.

SAMP lyase and AMP deaminase were determined in parenchymal and kupffer cells of rats fed either basal or carcinogen-enriched diets. Results were calculated on a U/mg protein and U/cell basis. Data indicated that although deaminase increased 1 1/2 to 2-fold in parenchymal cells on a U/mg protein and U/cell basis from rats fed carcinogen-enriched diets there was a greater increase in U/mg protein. In contrast, little to no increase was seen in kupffer cells. SAMP lyase, however, depicted a smaller increase in parenchymal cells of carcinogen-enriched diet fed rats, but a 4- to 5-fold elevation in kupffer cells regardless of whether the data were expressed in U/mg protein or U/cell. These data indicate that increased activity of AMP deaminase may be a result of resistance to degradation in parenchymal cells, whereas SAMP lyase elevations in kupffer cells may reflect an increase in enzyme concentration.

AMP Deaminase↗

Ultraviolet irradiation of Rana pipiens embryos delays the migration of primordial germ cells into the genital ridges.

Ultraviolet (UV) irradiation of the vegetal pole of anuran embryos at the two-cell stage has been reported to cause aberrant cleavage as well as a subsequent reduction in germ cell numbers. In this study, we find no correlation between UV-induced cleavage abnormalities and the absence of primordial germ cells in Rana pipiens tadpoles examined at stage 25. On the other hand, some tadpoles from a population which was lacking primordial germ cells at stage 25 subsequently contained germ cells. These late-appearing germs cells exhibited damaged mitochondria, autophagosomes, and secondary lysosomes, while surrounding somatic cells were morphologically normal. We suggest that these cytoplasmic abnormalities resulted from an effect of the initial UV irradiation of germ plasm. We conclude that one effect of UV irradiation of germ plasm is to delay or inhibit the migration of primordial germ cells into the genital ridges.

Animals↗

Developmentally regulated RNA binding proteins during oogenesis in Xenopus laevis.

During oogenesis, Xenopus oocytes synthesize and accumulate all types of RNA. In particular, they store poly(A+) RNA to such an extent that only about 5% is actually translated in the oocyte. Using a protein blotting and in vitro binding assay, we have identified proteins which are associated with poly(A+) RNA and perhaps other RNAs as well. Two groups of binding proteins were identified. The first group accumulates during oogenesis, generally is less than 50,000 molecular weight, and sediments in the 80 S and polysome regions of a gradient. These proteins most likely include ribosomal proteins. A second group of proteins is oocyte-specific, sediments less than 80 S as well 80 S and slightly heavier, generally has molecular weights greater than 50,000, and diminishes in amount as oogenesis progresses. In addition, these proteins are retained by oligo(dT)-cellulose when ribonucleoproteins are analyzed by chromatography and, when challenged with several different types of RNA in vitro, bind poly(A+) RNA preferentially. The possibility that some of these proteins might regulate the stability or translatability of mRNAs during oogenesis is discussed.

Animals↗

The recruitment of membrane-bound mRNAs for translation in microinjected Xenopus oocytes.

Xenopus oocytes were injected with several heterologous mRNAs to investigate specific message recruitment onto polysomes. When large amounts of mRNA such as ovalbumin, which is translated on membrane-bound polysomes, are injected, most of the stable message accumulates in a postpolysomal supernatant. Conversely, when similar amounts of nonmembrane-bound mRNAs such as those which code for adenovirus protein are injected, most of the messages are found in a polysome pellet. Nonpolysomal mRNAs such as zein, ovalbumin, and lysozyme can be recruited onto membrane-bound polysomes by injected rough endoplasmic reticulum (RER). Only RER, but not Golgi apparatus, plasma membrane, or salt-washed RER has such recruiting ability. In addition, the nontranslating messengers can be recruited by injecting the salt wash of the RER. Since most nontranslating mRNAs sediment to regions less than the 80 S monosome, the step at which translational regulation takes place is probably initiation rather than elongation. The mechanism by which recruitment might occur is discussed.

Adenoviridae↗

Internal pH of Xenopus oocytes: a study of the mechanism and role of pH changes during meiotic maturation.

The internal pH (pHi) of Xenopus laevis oocytes, as measured by the DMO method, covered a broad range of values from 7.06 +/- 0.01 to 7.93 +/- 0.01, with a mean value of 7.43 +/- 0.03. The pHi measured by DMO and microelectrodes was nearly identical in control and maturing oocytes from the same batch. The oocytes from most females elevated their pHi in response to progesterone, reaching a maximum elevation of 0.30 +/- 0.03 pH units above control values at 100% germinal vesicle breakdown (GVBD). However, some females were found to contain oocytes that already had an elevated pHi of 7.71 +/- 0.03 which did not significantly increase during maturation. Human chorionic gonadotrophin (hcG)-stimulated females had oocytes with slightly higher control pHi values than oocytes from nonstimulated females but still showed the same elevation in response to progesterone. Thus, the "stimulated" state of oocyte physiology as induced by hcG did not account for the variation in control pHi and responsiveness to progesterone. Other aspects of this variability are discussed. Elevating or lowering the external pH is shown to elevate and lower pHi, respectively, in a stable and predictable manner. Using this approach to change pHi we have found no effect of changes in pHi on the rate of protein synthesis in control and maturing oocytes. Similarly, pHi had only a slight facilitating effect on the rate of GVBD. A pH indicator gel was used to demonstrate that the pHi increase during oocyte maturation involved an acid efflux. We conclude that an elevated pHi is not necessary for oocyte maturation, yet the mechanism of the pHi elevation is discussed as a possible lead to events that are necessary.

Animals↗

RNA stabilization and continued RNA processing following nuclear dissolution in maturing Xenopus laevis oocytes.

The Xenopus laevis oocyte provides a convenient system for studying cell cycle-induced changes in RNA metabolism. Initially arrested at first meiotic prophase, it can be induced in vitro to undergo maturation by incubation in the presence of progesterone, progressing to second meiotic metaphase over the course of several hours. The dissolution of the nuclear membrane before the onset of first meiotic metaphase (also known as germinal vesicle breakdown, or GVBD) provides a convenient indication of the overall rate of maturation. It is reported that all ordinarily unstable classes of nuclear RNA show substantial increases in stability following GVBD. However, some radioactivity in rRNA precursors disappears following GVBD and this is quantitatively accounted for by the appearance of label in mature rRNA. These results suggest continued processing of rRNA precursors after GVBD.

Animals↗

Regulation of adenovirus transcription by an E1a gene in microinjected Xenopus laevis oocytes.

The regulation of adenovirus type 5 gene expression by the E1a gene product was examined in microinjected Xenopus laevis oocytes. Chimeric genes were constructed which included the promoter region of early adenovirus type 5 gene 3 and the structural sequence which codes for the bacterial enzyme chloramphenicol-3-O-acetyltransferase (CAT). A plasmid containing this chimeric gene as well as plasmids containing the E1a gene were coinjected into oocyte nuclei. The presence of the E1a gene was shown to increase CAT activity by up to 8.5-fold over basal levels. Synthesis of the functional product from the E1a gene requires the removal of intron sequences by RNA splicing. The E1a gene and a derivative that precisely lacks the intron were equally effective in increasing CAT activity, suggesting that splicing of the primary E1a transcript is efficiently accomplished in the oocyte nucleus. This was confirmed by directly examining the E1a mRNAs by the S1 mapping procedure. A protein extract from adenovirus type 5-infected HeLa cells enriched for the E1a protein may supplant the E1a plasmid in enhancing CAT activity. Synthesis of the CAT enzyme after gene injection is invariant in oocytes from the same frog, but oocytes from different frogs show a high degree of variability in their ability to synthesize the CAT enzyme. Microinjected X. laevis oocytes appear to be an extremely useful system to study the effects of protein elements on transcription.

Acetyltransferases↗

A comparison of hepatic adenylosuccinate lyase from rats fed either a chow diet or a semisynthetic basal diet low in riboflavin.

1. AMPS Lyase which shows increased activity in both transplantable and primary hepatomas has been purified approximately 400-fold from both chow fed and basal fed rat liver. 2. The enzyme from basal fed rats shows subtle but significant differences from the enzyme from chow fed rats. Especially noted was the difference in effect of 100 mM Na+ on the enzyme from the two different sources after the whole liver had been frozen. 3. These differences may be related to the effects of starvation followed by refeeding or administration of either glucose or corn oil.

Adenylosuccinate Lyase↗

Stimulation of Xenopus oocyte protein synthesis by microinjected adenovirus RNA.

The ability of injected heterologous mRNAs to compete with endogenous mRNAs in Xenopus oocytes was assayed. In confirmation of previous reports, globin mRNA translation in oocytes results in a concomitant decrease in endogenous protein synthesis. In contrast, injection of adenovirus 5 mRNA into the oocyte results in a stimulation of endogenous protein synthesis. The stimulation is dose dependent and does not require nuclear transcription in the oocyte. Preliminary mapping data suggest that the stimulatory RNA is a product of one of the viral immediate early genes.

Adenoviruses, Human↗

Differential capacity for translation and lack of competition between mRNAs that segregate to free and membrane-bound polysomes.

The Xenopus oocyte was used to study translation, stability and subcellular compartmentation of injected heterologous mRNAs. The capacity of the oocyte to translate a membrane-free mRNA, globin, was not saturable by amounts up to 100 ng injected. In contrast, the ability of the oocyte to translate a membrane-associated mRNA, zein, saturated at 20 ng or less of injected mRNA. The hybridization of RNA from message-injected oocytes to cDNA probes revealed that approximately the same amount of globin and zein mRNAs remained stable and that this fraction was as little as 2.1% at the highest amount injected (80 ng). However, while almost all of the globin mRNA at all concentrations injected was found in a polysome pellet, most of the zein mRNA at saturating concentrations was found in a post-polysomal supernatant. The reason for the limited translation of zein mRNA appears to be due to a paucity of oocyte rough endoplasmic reticulum since the injection of dog pancreas rough endoplasmic reticulum into oocytes previously injected with zein mRNA stimulates zein synthesis. Displacement and competition experiments between four different mRNAs indicate that injected messages are distributed to two translational compartments, membrane-bound and free, and that this occurs either prior to the initiation of translation or involves message-specific factors.

Animals↗