Thylakoid membrane architecture

Confocal scanning laser microscopic observations were made on live chloroplasts in intact cells and on mechanically isolated, intact chloroplasts. Chlorophyll fluorescence was imaged to observe thylakoid membrane architecture. C-3 plant species studied included Spinacia oleracea L., Spathiphyllum sp. Schott, cv. 'Mauna Loa', and Pisum sativum L. C-4 plants were also investigated: Saccharum officinarum L., Sorghum bicolor L. Moench, Zea mays L. and Panicum miliaceum L. Some Spinacia chloroplasts were treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to enhance or sodium dithionite (SD) to reduce the photosystem II fluorescence signal. Confocal microscopy images of C-3 chloroplasts differed from electron microscopy pictures because they showed discrete spots of bright fluorescence with black regions between them. There was no evidence of fluorescence from stroma thylakoids. The thylakoid membrane system at times appeared to be string-like, with brightly fluorescing grana lined up like beads. C-4 bundle sheath chloroplasts were imaged from three different types of C-4 plants. Saccharum and Sorghum bundle sheath chloroplasts showed homogeneous fluorescence and were much dimmer than mesophyll chloroplasts. Zea had rudimentary grana, and dim, homogeneous intergrana fluorescence was visualised. Panicum contained thylakoids similar in appearance and string-like arrangement to mesophyll chloroplasts. Isolated Pisum chloroplasts, treated with a drop of 5 mM MgCl2 showed a thylakoid membrane system which appeared to be unravelling. Spongy mesophyll chloroplasts of Spinacia treated with 5 mM sodium dithionite showed a granal thylakoid system with distinct regions of no fluorescence. A time-series experiment provided evidence of dynamic membrane rearrangements over a period of half an hour.

Extraordinary and extensive karyotypic variation: A 48-fold range in chromosome number in the gall-inducing scale insect Apiomorpha (Hemiptera : Coccoidea : Eriococcidae)

Chromosome number reflects strong constraints on karyotype evolution, unescaped by the majority of animal taxa. Although there is commonly chromosomal polymorphism among closely related taxa, very large differences in chromosome number are rare. This study reports one of the most extensive chromosomal ranges yet reported for an animal genus. Apiomorpha Rubsaamen (Hemiptera: Coccoidea: Eriococcidae), an endemic Australian gall-inducing scale insect genus, exhibits an extraordinary 48-fold variation in chromosome number with diploid numbers ranging from 4 to about 192. Diploid complements of all other eriococcids examined to date range only from 6 to 28. Closely related species of Apiomorpha usually have very different karyotypes, to the extent that the variation within some species- groups is as great as that across the entire genus. There is extensive chromosomal variation among populations within 17 of the morphologically defined species of Apiomorpha indicating the existence of cryptic species-complexes. The extent and pattern of karyotypic variation suggests rapid chromosomal evolution via fissions and (or) fusions. It is hypothesized that chromosomal rearrangements in Apiomorpha species may be associated with these insects' tracking the radiation of their speciose host genus, Eucalyptus.

Extensive chromosomal variation associated with taxon divergence and host specificity in the gall-inducing scale insect Apiomorpha munita (Schrader) (Hemiptera : Sternorrhyncha : Coccoidea : Eriococcidae)

Apiomorpha Rubsaamen (Hemiptera: Coccoidea: Eriococcidae) is one of the most chromosomally diverse of all animal genera. There is extensive karyotypic variation within many of the morphologically defined species, including A. munita (Schrader) which is here reported to have diploid chromosome counts ranging from 6 to more than 100. Each of the three morphologically defined subspecies of A. munita also displays considerable chromosomal variation: A. m. tereticornuta Gullan (2n =6, 8, 20, 22 or 24), A. m. malleensis Gullan (2n =6, 20, 22, 24 or 26), and A. m. munita (Schrader) (2n=54 or >100). Apiomorpha munita appears to occur only on eucalypts of the informal subgenus Symphyomyrtus, with each of the subspecies of A. munita restricted to discrete symphyomyrt sections. Several different karyotypic forms within each subspecies of A. munita appear to be restricted to only one or a few eucalypt species or series. The association between apparent host specificity and chromosomal rearrangements in A. munita suggests that both may be playing an active role in taxon divergence in Apiomorpha. (C) 2001 The Linnean Society of London.

Review of genetic and epigenetic alterations in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is associated with multiple risk factors and is believed to arise from pre-neoplastic lesions, usually in the background of cirrhosis. However, the genetic and epigenetic events of hepatocarcinogenesis are relatively poorly understood. HCC display gross genomic alterations, including chromosomal instability (CIN), CpG island methylation, DNA rearrangements associated with hepatitis B virus (HBV) DNA integration, DNA hypomethylation and, to a lesser degree, microsatellite instability. Various studies have reported CIN at chromosomal regions, 1p, 4q, 5q, 6q, 8p, 10q, 11p, 16p, 16q, 17p and 22q. Frequent promoter hypermethylation and subsequent loss of protein expression has also been demonstrated in HCC at tumor suppressor gene (TSG), p16, p14, p15, SOCS1, RIZ1, E-cadherin and 14-3-3 sigma. An interesting observation emerging from these studies is the presence of a methylator phenotype in hepatocarcinogenesis, although it does not seem advantageous to have high levels of microsatellite instability. Methylation also appears to be an early event, suggesting that this may precede cirrhosis. However, these genes have been studied in isolation and global studies of methylator phenotype are required to assess the significance of epigenetic silencing in hepatocarcinogenesis. Based on previous data there are obvious fundamental differences in the mechanisms of hepatic carcinogenesis, with at least two distinct mechanisms of malignant transformation in the liver, related to CIN and CpG island methylation. The reason for these differences and the relative importance of these mechanisms are not clear but likely relate to the etiopathogenesis of HCC. Defining these broad mechanisms is a necessary prelude to determine the timing of events in malignant transformation of the liver and to investigate the role of known risk factors for HCC.

The mechanisms of combustion and continuous reactions during mechanical alloying

Some materials exhibit a combustion event during mechanical alloying, which results in the rapid transformation of reactants into products, while others show a slow transformation of reactants into products, In this paper, the continuous W + C --> WC reaction is compared to the Ti + C --> TiC combustion reaction. Rietveld refinement of X-ray diffraction patterns is used to show that these particular reactions proceed through different pathways, determined by crystallographic factors of the reactants. When a crystallographic relationship exists between the reactants and the products, such as that between W and WC, the product forms slowly over a period of time. In contrast, insertion of C into the Ti structure is associated with atomic rearrangements within the crowded lattice planes and the subsequent catastrophic failure of the reactant lattices results in combustion to form TiC. (C) 2001 Academic Press.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

2-pyrazinylnitrene and 4-pyrimidylnitrene. Ring expansion to 1,3,5-triazacyclohepta-1,2,4,6-tetraene and ring opening to (2-isocyanovinyl)carbodiimide

Tetrazolo[1,5-a]pyrazine/2-azidopyrazine 9T/9A undergo photolysis in Ar matrix at cryogenic temperatures to yield 1,3,5-triazacyclohepta-1,2,4,6-tetraene 21 as the first observable intermediate, and 1-cyanoimidazole 11 and (2-isocyanovinyl)carbodiimide 22 as the final products. The latter tautomerizes to 2-(isocyanovinyl)cyanamide 23 on warming to 40 K. The same intermediate 21 and the same final products are obtained on matrix photolysis of the isomeric tetrazolo[1,5-c]pyrimidine/4-azidopyrimidine 24T/24A. These photolysis results as well as those of the previously reported thermal ring contraction of N-15-labeled 2-pyrazinyl- and 4-pyrimidylnitrenes to 1-cyanoimidazoles can all be rationalized in terms of selective ring opening of 21 or nitrine 10 to a nitrile ylide zwitterion 28 prior to formation of the final products, 11 and 22. The results are supported by high-level ab initio and DFT calculations (CASPT2-CASSCF(6,6), G3(MP2), and B3LYP/6-31+G*) of the energies and IR spectra of the intermediates and products.

2-cyano(2-pyridyl)ketene

Flash vacuum thermolysis of quinolizinones is a new way of generating ketenes. The title ketene is obtained from 1-cyano-2-hydroxyquinolizine-4-one and characterized by its Ar matrix infrared spectrum. (C) Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany 2002.

Germline BRCA1 promoter deletions in UK and Australian familial breast cancer patients: Identification of a novel deletion consistent with BRCA1 :psi VBRCA1 recombination

Inherited susceptibility to breast cancer results from germline mutations in one of a number of genes including BRCA1. A significant number of BRCA1-linked familial breast cancer patients, however, have no detectable BRCA1 mutation. This could be due in part to the inability of commonly used mutation-detection techniques to identify mutations outside the BRCA1 coding region. This paper addresses the hypothesis that non coding region mutations, specifically in the BRCA1 promoter, account for some of these cases. We describe a new and detailed restriction map of the 5' region of the BRCA1 gene including the nearby NBR2, psiBRCA1, and NBR1 genes and the isolation of a number of new informative hybridization probes suitable for Southern analysis. Using this information we screened DNA from lymphoblastoid cell-lines made from 114 UK familial breast cancer patients and detected one large deletion in the 5' region of BRCA1. We show that the breakpoints for this deletion are in BRCA1 intron 2 and between NBR2 and exon 2 of psiBRCA1, raising the possibility that this deletion arose via a novel mechanism involving BRCA1:psiBRCA1 recombination. We have also screened 60 familial breast cancer patients from the Australian population, using an amplification refractory mutation system (ARMS) technique described previously by our group, and found one patient with a genotype consistent with a BRCA1 promoter deletion. These findings indicate that germline BRCA1 promoter deletions are a rare and yet significant mutation event and that they could arise via a novel genetic mechanism. Hum Mutat 19:435-442, 2002. (C) 2002 Wiley-Liss, Inc.

Characterization of the mouse hnRNP A2/B1/B0 gene and identification of processed pseudogenes

The mouse hnRNP A2/B1/B0 gene has been cloned using a PCR-based strategy and sequenced. Analysis of this sequence showed that the gene organization closely follows that of the human orthologue with 12 exons and 11 introns. The hnRNP A2/B1/B0 gene gives rise to four splice variants through alternative splicing of exons 2 and 9. RT-PCR assays indicated that all splice variants were expressed in mouse brain, skin, and stomach tissues of varying ages, although their ratios to one another varied with age and tissue type. We also identified a small subset of all polyadenylated splice variants that included intron 11, which shows 94% sequence identity between human and mouse. Several processed pseudogenes were identified in the mouse genome. A search of the mouse genome databases located five pseudogenes, four of. which are presumed to be non-functional because of the presence of premature stop codons, large deletions or rearrangements within the coding region. The fifth, which possesses putative promoter elements and has a coding sequence identical to that of the hnRNP A2 mRNA, variant, may be functional. (C) 2002 Elsevier Science B.V. All rights reserved.

Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor

The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R,) that confers agonist selectivity for Are in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L-296, N-297, and R-298. A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.

The value of idiosyncratic markers and changes to conserved tRNA sequences from the mitochondrial genome of hard ticks (Acari: Ixodida: Ixodidae) for phylogenetic inference

Idiosyncratic markers are features of genes and genomes that are so unusual that it is unlikely that they evolved more than once in a lineage of organisms. Here we explore further the potential of idiosyncratic markers and changes to typically conserved tRNA sequences for phylogenetic inference. Hard ticks were chosen as the model group because their phylogeny has been studied extensively. Fifty-eight candidate markers from hard ticks ( family Ixodidae) and 22 markers from the subfamily Rhipicephalinae sensu lato were mapped onto phylogenies of these groups. Two of the most interesting markers, features of the secondary structure of two different tRNAs, gave strong support to the hypothesis that species of the Prostriata ( Ixodes spp.) are monophyletic. Previous analyses of genes and morphology did not strongly support this relationship, instead suggesting that the Prostriata is paraphyletic with respect to the Metastriata ( the rest of the hard ticks). Parallel or convergent evolution was not found in the arrangements of mitochondrial genes in ticks nor were there any reversals to the ancestral arthropod character state. Many of the markers identified were phylogenetically informative, whereas others should be informative with study of additional taxa. Idiosyncratic markers and changes to typically conserved nucleotides in tRNAs that are phylogenetically informative were common in this data set, and thus these types of markers might be found in other organisms.

3-pyridylcarbene and 3-pyridylnitrene: Ring opening to nitrile ylides

Photolysis of 3-pyridyldiazomethane in an Ar matrix at 7-10 K gives 3-pyridylcarbene. Further photolysis causes ring opening to nitrile ylide 26 (formonitrile pent-2-en-4-ynylide) as the major reaction together with a minor amount of ring expansion to 1-azacyclohepta-1,3,4,6-tetraene, 27. Matrix photolysis of 3-azidopyridine leads to ring opening to formonitrile N-cyanovinylmethylide, 33.

2-quinolyl- and 1-isoquinolylnitrenes: Ring expansion and ring opening in heteroarylnitrenes

Argon matrix photolysis of tetrazolo[1,5-a]quinoline 8 and tetrazolo[5,1-a]isoquinoline 7 causes nitrogen elimination and ring expansion to 1,3-diazabenzo[d]cyclohepta-1,2,4,6-tetraene 13. The photolysis of tetrazolo[5,1-a]isoquinoline 7 also causes ring opening to o-cyanophenylketenimine 22. Mechanisms of ring opening of heteroarylnitrenes are discussed.

Prenatal diagnosis of tetrasomy 18p using multiplex fluorescent PCR and comparison with a variety of techniques

We report genetic characterization of isochromosome 18p using a combination of cytogenetic and molecular genetic methods, including multiplex fluorescent PCR. The patient was referred for chorionic villus sampling (CVS) due to advanced maternal age and maternal anxiety. The placental karyotype was 47,XX,+mar, with the marker having the appearance of a small supernumerary isochromosome. Because differentiating between isochromosomes and other structural rearrangements is normally very difficult, a variety of genetic tests including fluorescence in situ hybridization (FISH), PCR, and multiplex fluorescent PCR were undertaken to determine chromosomal origin and copy number and, thus, allow accurate diagnosis of the corresponding syndrome. FISH determined that the marker chromosome contained chromosome 18 material. PCR of a variety of short tandem repeats (STRs) confirmed that there was at least one extra copy of the maternal 18p material. However, neither FISH nor PCR could accurately determine copy number. Multiplex fluorescent PCR (MF-PCR) of STRs simultaneously determined that: (1) the marker included 18p material; (2) the marker was maternal in origin; (3) allele copy number indicated tetrasomy; and (4) contamination of the sample could be ruled out. Results were also rapid with accurate diagnosis of the syndrome tetrasomy 18p possible within 5 hours.