Production of membrane whorls in rat liver by some inhibitors of protein synthesis.

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [(3)H]leucine and of [(3)H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [(3)H]leucine incorporation into cytoplasmic membranes was inhibited, while [(3)H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.

Hepatic organelle interaction. IV. Mechanism of succinate enhancement of formaldehyde accumulation from endoplasmic reticulum N-dealkylations.

Further evidence for organelle interaction during drug metabolism by the liver is presented. The apparent stimulation by succinate of formaldehyde accumulation in the medium, which was reported to occur with liver slices and homogenates as well as with mitochondria plus microsomes, has been shown to be the result of succinate inhibition of mitochondrial aldehyde dehydrogenase. The mechanism of succinate inhibition is shown to be by reverse electron transport, and an increase in the NADH to NAD+ ratio in the mitochondria; the aldehyde dehydrogenase requires the oxidized form of the pyridine nucleotide as its cofactor. Studies on in vitro N-demethylation by liver microsomes and endoplasmic reticulum segments which cosediment with the mitochondria indicate that formaldehyde produced by the mixed function oxidase is handled differently from formaldehyde added to the medium. The latter is mainly retained in the medium containing 5 mM semicarbazide, while the generated formaldehyde is more than 50% consumed by the mitochondria. Electron microscopy has indicated that the microsomes and the endoplasmic reticulum fragments have a tendency to align themselves close to the mitochondria when present in the same medium. Consequently, it is possible that formaldehyde released to the medium adjacent to the mitochondria, as by N-demethylation, would be exposed to semicarbazide for shorter periods than that added directly to the medium. In agreement with this suggestion, complexing of formaldehyde with semicarbazide was observed spectroscopically not to be an extremely rapid reaction even at 37 degrees C. This is believed to be the reason for the greater extent of consumption of formaldehyde generated by the endoplasmic reticulum.

Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila.

Members of the myocyte enhancer binding factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, and serum response factor) box transcription factors are expressed in the skeletal, cardiac, and smooth muscle lineages of vertebrate and Drosophila embryos. These factors bind an adenine-thymidine-rich DNA sequence associated with muscle-specific genes. The function of MEF2 was determined by generating a loss-of-function of the single mef2 gene in Drosophila (D-mef2). In loss-of-function embryos, somatic, cardiac, and visceral muscle cells did not differentiate, but myoblasts were normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2.

Sequence and functional properties of Ets genes in the model organism Drosophila.

Detailed molecular and genetic studies, coupled with the recent sequencing of the fly genome, have identified eight Ets-related genes in the model organism Drosophila. All show homology to genes in vertebrate species. Functional analyses of some of the Drosophila ets genes have revealed their essential roles in developmental processes such as metamorphosis, oogenesis, neurogenesis, myogenesis, and eye development. Such studies have yielded important insights into our understanding of the genetic control of hormonally-regulated gene expression, programmed cell death, and signal transduction during cell fate determination and differentiation. The developmental roles of E74 (ELF1), pointed (Ets 1), yan (TEL), and D-elg (GABPalpha) will be reviewed in this article. The context of their participation in signal transduction and gene regulation will also be discussed. The information should be of significant value to the study of related processes in higher organisms due to the growing evidence for the cross species conservation of developmental mechanisms.

Requirement of the ETS domain transcription factor D-ELG for egg chamber patterning and development during Drosophila oogenesis.

The D-elg gene encodes an ETS domain transcription factor that functions in Drosophila oogenesis. D-elg belongs to a small group of genes that are required for the formation of both the anteroposterior and dorsoventral axes of the egg chamber. During oogenesis in D-elg mutant females, the spatial localization of oskar and gurken mRNAs in the oocyte is disrupted and a follicle cell enhancer trap marker identifies dorsoventral polarity defects. Also, specialized follicle cells, called border cells, fail to migrate from their anterior location to a position adjacent to the developing oocyte. Consistent with these phenotypes, D-elg shows genetic interactions with two genes required for normal egg chamber differentiation.

Ventral neuroblasts and the heartless FGF receptor are required for muscle founder cell specification in Drosophila.

Muscle founder cells are uniquely specified cells that fuse with neighboring myoblasts to generate the complex pattern of body wall muscles in the Drosophila embryo. We have investigated the positional specification of founder cells for ventral oblique muscles, marked by the restricted expression of tinman RNA and the activity of a D-mef2 enhancer. The formation of these ventral myoblasts requires the function of the Heartless FGF receptor in the mesoderm and the presence of ventral neuroblasts in the central nervous system. Overproduction of ventral neuroblasts due to the forced expression of the homeodomain protein Vnd leads to increased numbers of founder cells. These results suggest the use of a neuroectoderm-to-mesoderm signaling pathway in the specification of ventral muscle precursors.

Characterization of lethal alleles of D-elg, an ets proto-oncogene related gene with multiple functions in Drosophila development.

We have used genetic complementation rescue to identify two lethal alleles of D-elg, an ets proto-oncogene related gene of Drosophila. Animals that are hemizygous or trans-heterozygous for the alleles die as pharate adults, demonstrating normal gene function is required to complete Drosophila development. Females trans-heterozygous for the 1(3)902 lethal allele and the previously characterized tne female sterile allele produce embryos with abdominal segmentation defects. This finding implicates a role for the D-elg gene in anterior-posterior patterning. The cloning and sequencing of the lethal alleles identified molecular mutations that may result in ELG protein truncation, altered ELG protein interactions, or defective D-elg mRNA splicing.

D-elg, a member of the Drosophila ets gene family: sequence, expression and evolutionary comparison.

We have cloned a cDNA from the Drosophila elg gene, a new member of the ets family of genes. The D-elg gene is located at 97D on chromosome 3R and is expressed as a 2.0kb RNA in the embryos, pupae and adults, with no detectable expression in third instar larvae. D-elg expression is observed in all cells of early stage embryos, prior to transcriptional activation of the zygotic genome, and is maintained throughout embryogenesis with no regional localization. The cDNA encodes a predicted protein of 15.4kD that has an 86 amino acid sequence with 72% similarity to the carboxy terminal region of the Drosophila ets-2 gene. Comparison with all known ets genes allows us to define a minimal region required for assignment to the ets gene family.

Molecular characterization and structural organization of D-elg, an ets proto-oncogene-related gene of Drosophila.

We have continued the molecular analysis of D-elg, a member of the Drosophila ets gene family. Based on the characterization of cDNA and genomic sequences, the D-elg gene contains five exons and four introns and produces a mRNA with an open reading frame of 464 amino acids. Consistent with this analysis, in vitro translation of a near full-length D-elg cRNA yields a protein with a molecular weight of approximately 56 kDa. D-elg shows significant homology to other ets proteins in the amino-terminal A domain and strong homology in the carboxy-terminal ETS domain. The D-elg protein is most similar to the alpha-subunit of the mouse GA-binding protein.

Expression of the D-MEF2 transcription in the Drosophila brain suggests a role in neuronal cell differentiation.

D-MEF2 is a MADS domain transcription factor expressed in the cardiac, somatic, and visceral muscle cell lineages in the Drosophila embryo. Genetic studies have demonstrated that D-mef2 gene function is required for the proper differentiation of all three of these muscle types. We show that D-MEF2 is also expressed in a limited number of other cells types during development, including Kenyon cells present in the mushroom bodies of the Drosophila brain. This finding suggests a role for D-mef2 in neuron differentiation. To investigate D-mef2 expression in muscle and Kenyon cells, we assayed 26 kb of D-mef2 5'-flanking and intragenic DNA for regulatory sequences controlling the expression of the gene. Our results show that separable enhancer sequences direct D-mef2 gene expression in the myogenic and neuronal cell lineages. The identification of these regulatory DNAs provides a starting point for the analysis of transcriptional regulators controlling the cell-specific expression of D-mef2 and a means to address the function of D-mef2 in Kenyon cell differentiation.

Diacridines: bifunctional intercalators. III. Definition of the general site of action.

Electron microscopy of HeLa cells exposed to spermine diacridine shows nucleolar distortions which disappear after several days despite the persistence of the metabolic changes promoted by spermine diacridine. This compound inhibits ribosomal RNA synthesis and appears to act independently of any particular phase of the cell cycle. The DNA content of the HeLa cells remains unchanged and the cell distribution is not significantly disturbed from its normal distribution in the various phases of the cell cycle. Spermine diacridine and other diacridines inhibit primarily chain initiation but also chain elongation by DNA-directed RNA polymerase of Azotobacter vinelandii.

Structure of the Eip28/29 gene, an ecdysone-inducible gene from Drosophila.

The EIPs 28 and 29 are a family of polypeptides identified originally by their ecdysone inducibility in Drosophila cell lines. At least two family members, 28III and 29III, appear to be primary translation products. Here we describe a unique Eip28/29 gene that must encode both primary products. The Eip28/29 gene is unique because the cloned genomic DNA hybridizes to both EIP 28 and 29 messenger RNAs under stringent conditions, but does not anneal detectably to other genomic sequences even under mild conditions. Furthermore the diverse products of this gene are not alleles because flies homozygous for the chromosomal region (71CD) containing the Eip28/29 gene produce mRNAs that translate to yield all the EIPs 28 and 29. We report here the sequence of a 2855-nucleotide region encompassing the Eip28/29 gene. By comparisons with complementary DNA sequences and by nuclease protection experiments we have derived a complete structure for the Eip28/29 transcription unit. The primary transcript is 2146 nucleotides long and is processed by the removal of three introns to yield the predominant mature transcript in tissue culture cells (979 nucleotides). This transcript probably corresponds to the 28III mRNA. Neither the start of the transcription unit nor the structure of the predominant transcript is affected by the hormone ecdysone. The genomic sequence reveals a series of heptanucleotide and octanucleotide repeats of unknown function that fall at about 50-nucleotide intervals within the first 150 nucleotides upstream from the transcription unit. In addition this sequence, when combined with previously published data, suggests that the consensus cap site sequence in Drosophila may be extended to include 13 nucleotides centered on the heptanucleotide core previously recognized by Snyder et al. (1982).

Conserved cardiogenic functions of the multitype zinc-finger proteins: U-shaped and FOG-2.

Multitype zinc-finger proteins murine Friend of GATA-2 (FOG-2) and Drosophila U-shaped (Ush) are required for heart development. Both FOG proteins participate in signal transduction pathways that are essential for cardiogenesis. FOG-2 regulates signaling from the myocardium, which is required for the production of the coronary vasculature. Ush functions in a common pathway with the Heartless (Htl) fibroblast growth factor (FGF) receptor to control mesodermal cell migration, which is required for cardiogenic cell fate commitment. In vitro studies have demonstrated that both FOG proteins repress GATA factor transcriptional activation of cardiac promoters. These similarities provide further evidence for the conservation of gene functions during cardiogenesis in Drosophila and higher eukaryotes.

The Drosophila melanogaster z600 gene encodes a chromatin-associated protein synthesized in the syncytial blastoderm.

The Drosophila melanogaster z600 gene is zygotically expressed with gene transcripts accumulating transiently during early embryogenesis. Based on nucleotide sequence analysis, z600 is predicted to encode a small, basic, histone-like protein. Antibodies generated against a beta-galactosidase::z600 fusion protein immunoprecipitated the z600 in vitro translation product and detected a z600 protein present predominantly in 2- to 4-h embryos. We localized the z600 protein in whole-amount embryos by indirect immunofluorescence. These studies show that the protein is located in the nucleus and associated with chromatin in the syncytial blastoderm.

The mechanism of actions of collagenase on the inhibition of release of acetylcholine from synaptosomal preparations.

The release of acetylcholine from synaptosomal preparations from bovine superior cervical ganglia and rat cortex was inhibited when the preparations were pretreated with collagenase. The inhibition of release could be overcome with the calcium ionophore A23187. Collagenase treatment was shown to inhibit the uptake of calcium into the preparations. In addition, gel electrophoresis of synaptosomal membranes revealed two missing high molecular weight proteins when either synaptosomes or synaptosomal membranes were incubated with collagenase.

View sampling requirements in fan beam computed tomography.

It is known that good CT reconstructions require that many views of the scanned object be obtained, where each view consists of many rays which traverse the patient. It is known that the required number of views depends on the diameter of the region reconstructed and the spatial resolution achieved. We present a new analysis of this question, valid for reconstruction from fan beam views, which shows that larger fan angles will require more views. Computer simulations, using a fan beam reconstruction algorithm, confirmed the predicted fan angle dependence and demonstrated that streak artifacts will result if too few views are used. The effect is also demonstrated experimentally by a phantom scan on a clinical CT scanner. Implications for clinical CT scanning are discussed.

An improved stereotactic technique for cyst cannulation.

Stereotactic techniques for cannulation of cystic structures, within the brain, are well known. Superimposed structures (vessels, ventricles, etc.) may make this problematic as does the need to approach the cystic structure perpendicular to its tangent plane (rather than "glancing") as with a craniopharyngioma cyst. To facilitate a three-dimensional visualization of the trajectory, we have employed digital holography. Transparent holographic images of cystic structures, ventricles, and sulci are rendered from T2-weighted MR data. Holographic images of vascular structures are rendered from CT or MR angiographic data. Vascular holograms are superimposed over the brain holograms, demonstrating the spatial relationships of these structures with regard to each other. Holographic images of the skull are rendered from CT slices. A Laitinen stereotactic frame (Sandstrom) is placed on the patient prior to obtaining the CT. The skull, pre-existing shunt catheters, and the stereotactic frame are all readily visible. The brain and vascular holograms are superimposed on these. The resulting image clearly demonstrates cystic structures, ventricles, vessels, pre-existing catheters, all within the skull and stereotactic frame. Using this holographic image as a "phantom", the actual Laitinen stereotactic frame is placed within its holographic image. The optical trajectory is then chosen, and the articulated arm of the stereotactic device is so adjusted. Subsequently, the frame is used to effect stereotactic placement of the cannula, in the usual manner. The major advantages of this technique are twofold. The first advantage lies with the fact that the surgeon can readily visualize the entire trajectory of the needle, and easily appreciate all structures which may be encountered by the needle on its passage from the skull to the target. Presumably, the surgeon's knowledge of anatomy would unable such knowledge to be apparent, but in complex cases the "safe" corridor may be rather small, and its limits may not be intuitively obvious. This is all the more the case, when obstacles along the pathway are pathologically distorted, or when they are not of tissue origin (shunt catheters, etc.). Employing this technique, we have successfully cannulated cystic structures in six patients, three of which presented with complex trajectory problems.

Robotic whole body stereotactic radiosurgery: clinical advantages of the Cyberknife integrated system.

Radiosurgery is defined as the delivery of high doses of ionising radiation, in mono- or hypo- fractionated treatments, to destroy tumours or focal areas of pathology. The clinical requirements of designing a radiosurgical treatment system include providing: a) a highly precise beam delivery to targets located throughout the body, b) a highly conformal dose distribution, c) the ability to irradiate both small and/or large complex-shaped lesions while minimising the dose to adjacent radiosensitive tissues and d) the ability to interactively track lesion motion due to normal patient motion. To accomplish this, the CyberKnife radiosurgery system has pioneered in this area by taking advantage of the inherent geometrical targeting precision of a commercial arm-based robotic system carrying a compact X-band linear accelerator and integrated with X-ray imaging and visualisation feedback systems. The arm-mounted linear accelerator, equipped with patient specific anatomical models, registered to the patient in real-time with image guidance, dynamically and safely delivers conformal and homogeneous radiation for therapeutic benefit. This paper details the components of the CyberKnife system and their integration in the clinical workflow of radiosurgery.