Exchange of protein molecules through connections between higher plant plastids.

Individual plastids of vascular plants have generally been considered to be discrete autonomous entities that do not directly communicate with each other. However, in transgenic plants in which the plastid stroma was labeled with green fluorescent protein (GFP), thin tubular projections emanated from individual plastids and sometimes connected to other plastids. Flow of GFP between interconnected plastids could be observed when a single plastid or an interconnecting plastid tubule was photobleached and the loss of green fluorescence by both plastids was seen. These tubules allow the exchange of molecules within an interplastid communication system, which may facilitate the coordination of plastid activities.

Tissue-Specific Protein Expression in Plant Mitochondria.

Although the physiological role of plant mitochondria is thought to vary in different tissues at progressive stages of development, there has been little documentation that the complement of mitochondrial proteins is altered in different plant organs. Because the phenomenon of cytoplasmic male sterility suggests an unusual function for mitochondria in floral buds, we examined the tissue-specific expression of mitochondrial proteins in petunia buds at several stages of development, using both fertile and cytoplasmic male sterile plants. On tissue prints of cryostat-sectioned buds, antibodies recognizing subunit A of the mitochondrial ATPase (ATPA) localized very differently from antibodies recognizing subunit II of the cytochrome oxidase (COXII), which indicated that mitochondria in the same tissue could differentially express mitochondrially encoded proteins. The petunia cytoplasmic male sterility-associated fused (pcf) gene encodes a protein that colocalized with ATPA and the nuclear-encoded mitochondrial alternative oxidase (AOA) in sporogenous tissues, where little COXII protein was found. These overlapping and differential localization patterns may provide clues to the molecular mechanism of cytoplasmic male sterility.

A heterologous maize rpoB editing site is recognized by transgenic tobacco chloroplasts.

Single nucleotides in plant chloroplast transcripts are edited from the genomically encoded C to U, often resulting in changes of the encoded protein sequence. Site-specific trans-acting factors are postulated to direct the selection of edited residues. In order to further define cis sequences required for RNA editing, we investigated whether two editing sites present in maize rpoB mRNA would be recognized by the editing machinery of transformed tobacco chloroplasts. A 93-nucleotide (nt) segment surrounding site I is sufficient to direct editing of the maize sequence in tobacco chloroplasts. However, an 86-nt segment surrounding maize site IV (which is genomically encoded as a T in tobacco) does not confer editing of this site, suggesting that trans-acting factors necessary for recognition of site IV are not present in tobacco. The maize sequences surrounding site I were found to compete with the endogenous rpoB for a depletable trans factor and to reduce editing of endogenous site I. The presence of exogenous maize site I was also found to decrease editing of endogenous tobacco site II, indicating that there is a shared aspect of editing for some closely spaced editing sites.

Editing of pre-mRNAs can occur before cis- and trans-splicing in Petunia mitochondria.

Plant mitochondrial mRNAs have recently been shown to undergo editing, involving cytidine-to-uridine changes relative to the DNA sequence. We have examined the temporal relationship of editing and intron removal in coxII mRNAs in Petunia mitochondria. By using differential hybridization to probes specific for edited and unedited RNA and by sequencing of individual unspliced coxII pre-mRNA cDNAs, we found that RNA editing at any editing site can precede the splicing event. Similar results were obtained from examinations of pre-mRNA cDNAs of nad1, a gene composed of multiple exons that are both cis and trans spliced. Thus, intron removal is not required before editing can occur. The existence of editing intermediates indicates that the editing process is not strictly coincident with transcription.

A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited.

RNA editing occurs in two higher-plant organelles, chloroplasts and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components.

Protein polymorphism generated by differential RNA editing of a plant mitochondrial rps12 gene.

The rps12 gene transcripts encoding mitochondrial ribosomal protein S12 are partially edited in petunia mitochondria. Different petunia lines were found vary in the extent of rps12 transcript editing. To test whether multiple forms of RPS12 proteins are produced in petunia mitochondria as a result of partial editing, we probed mitochondrial proteins with specific antibodies against edited and unedited forms of a 13-amino-acid RPS12 peptide spanning two amino acids affected by RNA editing. Both antibodies reacted with mitochondrial proteins at the expected size for RPS12 proteins. The amounts of unedited RPS12 protein in different petunia lines correlate with the abundance of unedited transcripts in these plants. Unedited rps12 translation products are also detected in other plant species, indicating that polymorphism in mitochondrial rps12 expression is widespread. Moreover, we show that RPS12 proteins recognized by both edited-specific and unedited-specific antibodies are present in a petunia mitochondrial ribosome fraction. These results demonstrate that partially edited transcripts can be translated and that the protein product can accumulate to detectable levels. Therefore, genes exhibiting incompletely edited transcripts can encode more than one gene product in plant mitochondria.

A single alteration 20 nt 5' to an editing target inhibits chloroplast RNA editing in vivo.

Transcripts of typical dicot plant plastid genes undergo C-->U RNA editing at approximately 30 locations, but there is no consensus sequence surrounding the C targets of editing. The cis-acting elements required for editing of the C located at tobacco rpoB editing site II were investigated by introducing translatable chimeric minigenes containing sequence -20 to +6 surrounding the C target of editing. When the -20 to +6 sequence specified by the homologous region present in the black pine chloroplast genome was incorporated, virtually no editing of the transcripts occurred in transgenic tobacco plastids. Nucleotides that differ between the black pine and tobacco sequence were tested for their role in C-->U editing by designing chimeric genes containing one or more of these divergent nucleotides. Surprisingly, the divergent nucleotide that had the strongest negative effect on editing of the minigene transcript was located -20 nt 5' to the C target of editing. Expression of transgene transcripts carrying the 27 nt sequence did not affect the editing extent of the endogenous rpoB transcripts, even though the chimeric transcripts were much more abundant than those of the endogenous gene. In plants carrying a 93 nt rpoB editing site sequence, transgene transcripts accumulated to a level three times greater than transgene transcripts in the plants carrying the 27 nt rpoB editing sites and resulted in editing of the endogenous transcripts from 100 to 50%. Both a lower affinity of the 27 nt site for a trans-acting factor and lower abundance of the transcript could explain why expression of minigene transcripts containing the 27 nt sequence did not affect endogenous editing.

Enhanced protein kinase B/Akt signalling in pituitary tumours.

Pituitary tumours have previously been shown to harbour several abnormalities that cause deregulation of the cell cycle, particularly down-regulation of expression of the cyclin-dependent kinase inhibitor p27. However, it has been unclear whether these are the primary initiating events, or are secondary to other more proximate alterations in signalling pathways. In other cellular systems the Akt signalling pathway has been associated with downstream modulation of cell-cycle control. The aim of the present study was to test the hypothesis that Akt signalling is enhanced in pituitary tumours, and to see if changes in Akt expression are related to previous findings on low expression levels of the nuclear cell-cycle inhibitor p27 in pituitary tumours. We examined normal and adenomatous human pituitary tissue for mRNA and protein expression of Akt1, Akt2 and p27, and the activation of Akt, as well the phosphatase involved in the inactivation of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). In pituitary adenomas Akt1 and Akt2 mRNA were found to be over-expressed compared with normal pituitary, while PTEN transcripts showed similar levels between the two tissue types. Immunohistochemical expression of phospho-Akt was found to be higher in the tumours than normal pituitaries, while the protein expression of nuclear p27 and PTEN was lower in the adenomas. However, the expression of p27 and Akt were not directly correlated. PTEN sequencing revealed no mutation in the coding region of the gene in pituitary adenomas, and thus we did not locate a cause for the increased phosphorylation of Akt. In summary, we have shown over-expression and activation of the Akt pathway in pituitary tumours, and we speculate that cell-cycle changes observed in such tumours are secondary to these more proximate alterations. Since Akt is a major downstream signalling molecule of growth factor-liganded tyrosine kinase receptors, our data are most compatible with an abnormality at this level as the primary driver of pituitary tumorigenesis.

Effects of erythromycin on membrane-bound chloroplast ribosomes from wild-type Chlamydomonas reinhardi and erythromycin-resistant mutants.

1. Treatment of wild-type cells of Chlamydomonas reinhardi with high concentrations of erythromycin results in increased recovery of membrane-bound chloroplast ribosomes, presumably by preventing polysomal runoff during harvesting of cells. No such membrane-retention effect is detected if erythromycin is added after harvesting of cultures, before cell breakage. 2. Growth of wild-type cells is inhibited by 10 microgram/ml erythromycin, but a concentration twice as high is required to increase recovery of membrane-bound wild-type ribosomes. On the other hand, the concentrations of erythromycin which inhibit growth of mutant ery-M1b produce a membrane-retention effect. Mutant ery-U1a is resistant to high concentrations of erythromycin and no membrane-retention effect is detectable at concentrations which produce one in wild type and ery-M1b. 3. These results can be reconciled by a two-point model of the mechanism of erythromycin action on chloroplast ribosomes in Chlamydomonas.

The male sterility-associated pcf gene and the normal atp9-1 gene in Petunia are located on different mitochondrial DNA molecules.

A mitochondrial DNA (mtDNA) region termed the S-pcf locus has previously been correlated with cytoplasmic male sterility (CMS) in Petunia. In order to understand the relationship of the S-pcf locus to homologous sequences found elsewhere in mtDNAs of both CMS and fertile lines, the structure of the mitochondrial genome of CMS Petunia line 3688 was determined by cosmid walking. The S-pcf locus, which includes the only copies of genes for NADH dehydrogenase subunit 3 (nad3) and small ribosomal subunit protein 12 (rps12) was found to be located on a circular map of 396 kb, while a second almost identical circular map of 407 kb carries the only copies of the genes for 18S and 5S rRNA (rrn18 and rrn5), the only copy of a conserved unidentified gene (orf25), and the only known functional copy of atp9. Three different copies of a recombination repeat were found in six genomic environments, predicting sub-genomic circles of 277, 266 and 130 kb. The ratio of atp9 to S-pcf mtDNA sequences was approximately 1.5 to 1, indicating that sub-genomic molecules carrying these genes differ in abundance. Comparison of the mtDNA organization of the CMS line with that of the master circle of fertile Petunia line 3704 reveals numerous changes in order and orientation of ten different sectors.

A functional mitochondrial ATP synthase proteolipid gene produced by recombination of parental genes in a petunia somatic hybrid.

A novel ATP synthase subunit 9 gene (atp9) was identified in the mitochondrial genome of a Petunia somatic hybrid line (13-133) which was produced from a fusion between Petunia lines 3688 and 3704. The novel gene was generated by intergenomic recombination between atp9 genes from the two parental plant lines. The entire atp9 coding region is represented on the recombinant gene. Comparison of gene sequences indicate that the 5' transcribed region is contributed by an atp9 gene from 3704 and the 3' transcribed region is contributed by an atp9 gene from 3688. The recombinant atp9 gene is transcriptionally active. The location of the 5' and 3' transcript termini are conserved with respect to the parental genes, resulting in the production of hybrid transcripts.

Mitochondrial DNA Sequence Divergence among Lycopersicon and Related Solanum Species.

Sequence divergence among the mitochondrial (mt) DNA of nine Lycopersicon and two closely related Solanum species was estimated using the shared fragment method. A portion of each mt genome was highlighted by probing total DNA with a series of plasmid clones containing mt-specific DNA fragments from Lycopersicon pennellii. A total of 660 fragments were compared. As calculated by the shared fragment method, sequence divergence among the mtDNAs ranged from 0.4% for the L. esculentum-L. esculentum var. cerasiforme pair to 2.7% for the Solanum rickii-L. pimpinellifolium and L. cheesmanii-L. chilense pairs. The mtDNA divergence is higher than that reported for Lycopersicon chloroplast (cp) DNA, which indicates that the DNAs of the two plant organelles are evolving at different rates. The percentages of shared fragments were used to construct a phenogram that illustrates the present-day relationships of the mtDNAs. The mtDNA-derived phenogram places L. hirsutum closer to L. esculentum than taxonomic and cpDNA comparisons. Further, the recent assignment of L. pennellii to the genus Lycopersicon is supported by the mtDNA analysis.

Erythromycin resistance and the chloroplast ribosome in Chlamydomonas reinhardi.

Five classes of erythromycin-resistant mutants of Chlamydomonas reinhardi have been identified. Each class corresponds to a different genetic locus, three nuclear and two chloroplast. The three nuclear loci appear to be unlinked, while Conde et al. (1975) have shown that the two chloroplast loci are linked, but not allelic. Mutants in each class have a unique pattern of cross-resistance in vivo to other antibiotics (lincomycin, carbomycin, and cleocin) that affect chloroplast protein synthesis. The chloroplast ribosomes from each class have a distinctive erythromycin-binding reaction in vitro.--Haploid and diploid strains containing combinations of different genes affecting the chloroplast ribosome were constructed to probe ribosome structure. New phenotypes were obtained by such combinations, demonstrating interactions between the gene products of a number of loci specifying ribosome components.

Edited transcripts compete with unedited mRNAs for trans-acting editing factors in higher plant chloroplasts.

Chloroplast RNA transcripts of vascular plants undergo C to U editing at approximately 30 sites, but there is no consensus sequence that identifies a C to be edited. Both sequences closely surrounding an edited C and unidentified site-specific trans-acting factors have been shown to be important for editing. The ability of an already edited transgenic sequence to bind and thus titrate a trans-acting editing factor was evaluated for two editing sites, ndhF and rpoB site 2. The U-containing rpoB transcripts did not affect editing of the endogenous rpoB transcripts, likely because the comparable C-containing transcripts containing 27 nucleotides surrounding the edited C were only 20% edited, indicating a low affinity of a trans-factor for this length of edited sequence. Surprisingly, U-containing ndhF transgene transcripts reduced endogenous ndhF transcript editing to the same degree as a C-containing transgene transcript. This indicates that the C target of editing is not a critical recognition feature for the site-specific trans-acting factor.

Amyotrophic lateral sclerosis. Recent advances in pathogenesis and therapeutic trials.

We reviewed the current status of pathogenesis and therapeutic trials in amyotrophic lateral sclerosis (ALS). Clinical studies have identified several rare but definable causes for apparent ALS. Certain clinical features previously considered unlikely to occur in ALS are found on careful examination. Epidemiologic surveillance and recent studies of neurotoxic plant seeds used in Guam have shed light on the pathogenesis of endemic ALS. Extensive analyses of biochemical, metabolic, immunologic, viral, and toxic factors have provided provocative results requiring further studies. Reflecting on some of these hypotheses, therapeutic trials have been performed more vigorously than ever. Amyotrophic lateral sclerosis is now investigated at the molecular genetic level. Human autopsy and experimental animal studies have expanded our understanding of basic mechanisms involving motoneuronal degeneration. In the future, we must continue a relentless search for the pathogenesis of ALS, prospective clinical studies to define the limits of ALS, and well-designed, controlled therapeutic trials.

Neuro-ophthalmologic complications of cardiac catheterization.

We examined ten patients who, from 1981 to 1986, sustained neuro-ophthalmologic events during cardiac catheterization. Eight patients, most of whom recovered, were believed to have sustained embolic phenomena. Two patients experienced a typical migraine during the catheterization and likewise did well. We conclude that the likelihood of sustaining a neuro-ophthalmic complication during cardiac catheterization is low and that the prognosis after having sustained such a complication is generally favorable. Evidence suggests that artery-to-artery emboli is the dominant pathogenic factor.

Amyotrophic lateral sclerosis: effects of acute intravenous and chronic subcutaneous administration of thyrotropin-releasing hormone in controlled trials.

We performed double-blind crossover trials to assess the effects of thyrotropin-releasing hormone (TRH) on amyotrophic lateral sclerosis patients. For acute intravenous trials, 500 mg TRH or placebo with norepinephrine was given at 1-week intervals (16 patients). CSF TRH concentration increased, and clinical side effects appeared with TRH. For chronic studies, 25 mg TRH and a saline placebo were given subcutaneously every day for 3 months (25 patients). CSF TRH level increased 29-fold after a single TRH injection, and mild transient side effects occurred. Vital signs, respiratory function, semiquantitative and quantitative neurologic function, muscle strength by manual and dynamometer testing, and EMG were studied. With daily TRH, 10 patients noted subjective improvement without objective evidence, and 10 patients complained of worsening of the disease with objective decline after TRH was stopped. Statistical analysis, however, showed no beneficial effects from either acute or chronic TRH trials.

Ischemic optic neuropathy: a complication of cardiopulmonary bypass surgery.

Ischemic optic neuropathy followed cardiopulmonary bypass surgery in the postoperative period in 7 of 7685 consecutive procedures. Th visual loss was unilateral in four patients and bilateral in three and there was little improvement. This ischemic infarction of the optic nerve disk was attributed to hypotension, hypothermia, and activation of certain complement factors by the bypass procedure.

Cryostat tissue printing: an improved method for histochemical and immunocytochemical localization in soft tissues.

A modification of a previously described tissue print technique has been developed in which soft tissues are frozen and sectioned in a cryostat prior to direct collection on nitrocellulose or nylon membranes. The inexpensive embedding technique described here allows accurate orientation of specimens prior to mounting, and mounted material may be stored easily after initial sectioning for future reexamination. Standard hand tissue prints of soft specimens exhibit tissue distortion and uneven delivery of material to the membrane, which limits resolution and makes interpretation difficult. Cryostat sections retain tissue fragments in their original arrangement relative to each other during printing and deliver a consistent and quantitative amount of material from all parts of the specimen. The cryostat tissue print technique is applied here to immature floral buds, demonstrating the tissue-level histochemical localization of beta-glucuronidase in transgenic plants and immunolocalization of a novel protein present only in mutant plants. This modified technique is applicable for examining both plant and animal tissues.

Radioimmunoassay and enzyme immunoassay methods for detecting viral hepatitis markers.

Enzyme immunoassay (EIA) and radioimmunoassay (RIA) methods for HBsAg were compared on the basis of responses to the AABB-CAP Viral Hepatitis Marker proficiency survey. Conversion of laboratories to the EIA method was documented for the period studied. Using standard incubation procedures, participants using the EIA method reported results comparable to those reported by laboratories using RIA methods. The short incubation procedures for RIA and EIA were both less sensitive and less specific than standard incubation procedures.