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PubMed2006

[Atrial flutter: an update].

Invasive electrophysiologic studies have changed the clinical outlook for patients with atrial flutter. Recognition of the reentrant circuit responsible for typical atrial flutter has led to the development of catheter ablation techniques that can prevent recurrence in >90% of cases. In addition, general understanding of atrial tachycardias has changed radically, such that ECG-based classifications are now obsolete. Atypical reentrant circuits associated with surgical scars or fibrotic areas in either atrium, which are indistinguishable from focal tachycardias on ECG, have been identified. These circuits also seem amenable to treatment by ablation. Recently, a new type of reentrant tachycardia that could be problematic in the future has emerged in patients who have undergone extensive left atrial ablation for the treatment of atrial fibrillation. These atypical circuits can be characterized using the mapping and entrainment techniques initially developed for typical flutter. In these cases, electroanatomical mapping, involving the construction of a virtual anatomical model of the atria, is extremely helpful. Despite the success of ablation, long-term prognosis is frequently overshadowed by the appearance of atrial fibrillation, which suggests that flutter and fibrillation share a common arrhythmogenic origin that is not modified by cavotricuspid isthmus ablation. In contrast with our clear electrophysiologic understanding of atrial flutter, little is known about the natural history of the condition because the literature has traditionally grouped patients with flutter and fibrillation together. Consequently, the complex relationship between the two arrhythmias has still to be clearly delineated. Primary prevention and preventing the development of atrial fibrillation after ablation remain outstanding clinical challenges.

Anisotropy↗
PubMed1992

Anomalous coronary arteries: angiographic findings in 80 patients.

Among 13010 adults who underwent coronary arteriography, 80 (0.61%) patients had a total of 83 anomalous coronary arteries. Thirty-three (41%) of the patients were of Hispanic origin, while out of the entire population studied 30% were Hispanic. The right coronary artery was the most common anomalous vessel. It was identified in 50 (62%) patients, arising in 35 from the left aortic sinus, in 14 from the posterior sinus, and in 1 from the left coronary artery. An anomalous circumflex artery was recognized in 22 (27%) patients. Nine (11%) patients presented an anomalous left anterior descending artery, 1 patient an anomalous left main coronary artery, and another an anomalous septal perforator artery. Twenty-three (29%) patients had concomitant congenital heart abnormalities, most commonly. bicuspid aortic valve and mitral valve prolapse. In each of 5 patients with complex congenital heart disease the course of the anomalous vessel could have interfered with a surgical procedure. In 4 cases anomalous coronary arteries were associated with either anomalous systemic venous circulation or anomalous cardiac veins. In 5 (6%) patients only, the anomalous coronary artery was solely responsible for a clinical event. Coronary atherosclerosis of the anomalous arteries was found in 28% of the patients, while the overall incidence of the disease in this series was 65%. Thus, anomalous coronary arteries are associated with a high incidence of congenital heart diseases, but do not appear to be associated with an increased risk for development of coronary atherosclerosis. The angiographic recognition of these vessels is important in patients who undergo coronary angioplasty or cardiac surgery. Variations in the frequency of congenital coronary anomalies as reported herein may be attributed to a genetic background.

Abnormalities, Multiple↗
PubMed1994

Purified yeast RNA polymerase II reads through intrinsic blocks to elongation in response to the yeast TFIIS analogue, P37.

Saccharomyces cerevisiae has a TFIIS-related transcription elongation factor, originally called P37 (Sawadogo, M., Sentenac, A., and Fromageot, P. (1979) J. Biol. Chem. 255, 12-15; Nakanishi, T., Nakano, A., Nomura, K., Sekimizu, K., and Natori, S. (1992) J. Biol. Chem. 267, 13200-13204), which binds directly to RNA polymerase II and stimulates read-through of intrinsic blocks to elongation. To elucidate functional features of this protein:protein interaction, we tested the ability of several forms of RNA polymerase II to respond to either full-length or an amino-terminal truncation of TFIIS. The variants of the polymerase differed in the structure of the carboxyl-terminal domain of the largest subunit or lacked two of the smaller subunits. No differences in ability to recognize intrinsic blocks to elongation or to read through them in response to either form of TFIIS were detected among these variants. Furthermore, ternary complexes containing each variant form of RNA polymerase cleave the 3' end of the nascent transcripts in response to TFIIS, a reaction previously reported for mammalian and Drosophila TFIIS (Kassavetis, G. A., and Geiduschek, E. P. (1993) Science 259, 944-945) and likely to be important in TFIIS function. Thus the carboxyl-terminal domain of the largest subunit and subunits four and seven of the polymerase, required in vivo, are not required in vitro for recognition of intrinsic blocks to elongation, read-through in response to TFIIS, or TFIIS-stimulated cleavage of the nascent transcript.

Base Sequence↗
PubMed2001

Infantile spasms in tuberous sclerosis complex.

The high incidence of infantile spasms (IS) and hypsarrhythmia in tuberous sclerosis complex (TSC) has long been emphasized but it is now clear that infants with TSC show clinical and EEG differences from those with classical West syndrome. Seizures at onset are mainly characterized by partial motor seizures and IS. Subtle partial seizures may be present in the early neonatal period and may precede the onset of IS. Visual recording techniques have led to significant progress in the classification of seizures associated with TSC, demonstrating that they have a focal or multifocal origin in the vast majority of cases. In most cases, an awake interictal EEG shows focal or independent multifocal spike and slow-wave activity at onset and later a pseudo-hypsarrhythmic pattern. Ictal EEG starts with focal spikes originating from the posterotemporal, or occipital regions followed by a generalized irregular slow transient and an abrupt diffuse flattening. Although the pathophysiological mechanisms responsible for the coexistence of partial seizures and IS are still unclear, IS associated with TSC may be the result of a rapid secondary generalization. The presence of IS due to TSC is strongly predicted by the cortical tuber count, while the age of onset of seizures and the age of occurrence of EEG foci depend on the localization of cortical tubers with an earlier expression of the parieto-occipital than of the frontal regions. Early recognition of these distinctive features appears worthwhile for therapeutic and prognostic implications. Despite the efficacy of vigabatrin the prognosis of IS is generally poor. Studies using combined topographic mapping of EEG, magnetic resonance imaging and positron emission tomography may provide new strategies for selecting candidates suitable for surgery.

Electroencephalography↗
PubMed2005

Biochemical and structural studies of A-to-I editing by tRNA:A34 deaminases at the wobble position of transfer RNA.

Initial RNA transcription produces several tRNAs (one in prokaryotes and plant chloroplasts and seven or eight in eukaryotes) that contain an adenosine (A) at the wobble position (position 34). However, in all cases, adenosine at position 34 is post-transcriptionally converted to inosine (I), producing mature tRNAs without adenosine at the wobble position. The enzymes responsible for this A-to-I conversion in tRNA are tadA (acting as a homodimer) in prokaryotes and the heterodimeric ADAT2-ADAT3 complex in eukaryotes. The genes encoding these proteins are essential for cell viability, illustrating the biological importance of A-to-I editing at the wobble position of tRNA. In this study, recombinant tadA proteins from Escherichia coli, Agrobacterium tumefaciens, and Aquifex aeolicus, as well as the ADAT2-ADAT3 proteins from Saccharomyces cerevisiae, were overexpressed in E. coli and purified to homogeneity by chromatography. Crystallization of a proteolytically cleaved A. tumefaciens tadA (missing the last eight amino acids at the C-terminus) produced high-quality crystals, and the structure was determined at 1.6 A resolution. In addition, enzymatic assays of the wild-type proteins as well as several mutants were carried out using both the full-length E. coli tRNA(arg2) and the truncated anticodon stem-loop motif as substrates. Our biochemical and structural studies, in combination with sequence and structural comparisons with other deaminases, allow us to propose a model of tadA-tRNA interaction that explains the molecular basis of tRNA recognition by tadA. In particular, a conserved FFxxxR motif at the C-terminus, which is unique to tadA, has been identified, and its critical role in tRNA substrate recognition is proposed. Furthermore, the structural study of prokaryotic tadA presented here also sheds light on tRNA substrate recognition and the possible evolutionary origin of the eukaryotic ADAT2-ADAT3 heterodimer.

Adenosine↗
PubMed1999

Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an in vitro melanoma model.

Increasing attention has been devoted to elucidating the mechanism of lost or decreased expression of MHC or melanoma-associated antigens (MAAs), which may lead to tumor escape from immune recognition. Loss of expression of HLA class I or MAA has, as an undisputed consequence, loss of recognition by HLA class I-restricted cytotoxic T cells (CTLs). However, the relevance of down-regulation remains in question in terms of frequency of occurrence. Moreover the functional significance of epitope down-regulation, defining the relationship between MHC/epitope density and CTL interactions, is a matter of controversy, particularly with regard to whether the noted variability of expression of MHC/epitope occurs within a range likely to affect target recognition by CTLs. In this study, bulk metastatic melanoma cell lines originated from 25 HLA-A*0201 patients were analyzed for expression of HLA-A2 and MAAs. HLA-A2 expression was heterogeneous and correlated with lysis by CTLs. Sensitivity to lysis was also independently affected by the amount of ligand available for binding at concentrations of 0.001 to 1 mM. Natural expression of MAA was variable, independent from the expression of HLA-A*0201, and a significant co-factor determining recognition of melanoma targets. Thus, the naturally occurring variation in the expression of MAA and/or HLA documented by our in vitro results modulates recognition of melanoma targets and may (i) partially explain CTL-target interactions in vitro and (ii) elucidate potential mechanisms for progressive escape of tumor cells from immune recognition in vivo.

Antibodies, Monoclonal↗
PubMed1999

Selection of cleavage site by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNAArg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12-N and 13-N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase.

Animals↗
PubMed2002

New artificial host compounds containing galactose end groups for binding chiral organic amine guests: chiral discrimination and their complex structures.

New linear host (1) and cyclic hosts (2 and 3), which have galactopyranose skeletons as chiral origins and oxyethylenes skeletons as binding sites, were designed based on the structural features extracted from the fructo-oligosaccharide derivatives, having a large chiral discrimination ability, and were then synthesized. These hosts showed chiral discrimination toward chiral organic ammonium salts. For example, the chiral discrimination ability (the ratio of association constants: K(R)/K(S)) of host 1, which has the highest value among them, was K(R)/K(S) = 3 for Trp-O-(i)Pr(+) and K(R)/K(S) = 0.7 for 1-(1-naphthyl)ethylammonium (NEA(+)) at 298 K in CHCl(3). It was clarified that host 1 changed the conformation from a linear structure to the pseudo-ring structure by complexation with cations such as alkali metallic ions and chiral organic ammonium ions. The (1)H NMR induced shifts of host 1 by adding the NEA(+) guests showed that the host-guest complex structures are clearly different, depending upon the chirality of the guest; in the complex with (R)-NEA(+), the naphthyl group of the guest is located above the oxyethylene skeleton of the host and in the complex with (S)-NEA(+), and the naphthyl group is located between the edges of the pseudo-ring of the host. The clearly different structure of the complex of host 1 with NEA(+) may be caused by the dynamic molecular recognition, thus the induced-fitting mechanism.

Binding Sites↗
PubMed2002

The epidemiology of autistic spectrum disorders: is the prevalence rising?

For decades after Kanner's original paper on the subject was published in 1943, autism was generally considered to be a rare condition with a prevalence of around 2-4 per 10,000 children. Then, studies carried out in the late 1990s and the present century reported annual rises in incidence of autism in pre-school children, based on age of diagnosis, and increases in the age-specific prevalence rates in children. Prevalence rates of up to 60 per 10,000 for autism and even more for the whole autistic spectrum were reported. Reasons for these increases are discussed. They include changes in diagnostic criteria, development of the concept of the wide autistic spectrum, different methods used in studies, growing awareness and knowledge among parents and professional workers and the development of specialist services, as well as the possibility of a true increase in numbers. Various environmental causes for a genuine rise in incidence have been suggested, including the triple vaccine for measles, mumps and rubella (MMR]. Not one of the possible environmental causes, including MMR, has been confirmed by independent scientific investigation, whereas there is strong evidence that complex genetic factors play a major role in etiology. The evidence suggests that the majority, if not all, of the reported rise in incidence and prevalence is due to changes in diagnostic criteria and increasing awareness and recognition of autistic spectrum disorders. Whether there is also a genuine rise in incidence remains an open question.

Age Distribution↗
PubMed2000

Lysosomal accumulation of oxidized phosphatidylcholine-apolipoprotein B complex in macrophages: intracellular fate of oxidized low density lipoprotein.

Oxidized phosphatidylcholine (OxPC) formed in oxidized low density lipoprotein (OxLDL) is thought to be involved in the development of atherosclerosis. OxPC has been found in foam cells in atherosclerotic lesions and suggested to be the epitope for OxLDL recognition by macrophages. OxPC is present as a complex with apolipoprotein B (apoB) in OxLDL, since some OxPC can bind with proteins. In the current study, the intracellular fate of OxPC-apoB complexes after internalization of OxLDL by macrophages was investigated. Murine macrophage cell line J774.1 was incubated with either OxLDL or acetylated LDL for 24 h, then the cells were further incubated for up to 24 h in new medium without lipoprotein. Modified apoB in the cells was quantitated by sandwich ELISA using monoclonal antibodies against OxPC and apoB. Intracellular OxLDL decreased rapidly for the first 4 h to approx. 20% of that before medium change, with the apparent metabolism of OxPC-apoB complex ceasing. OxPC-apoB complexes that remained in the cells after 24 h chasing increased as the period of OxLDL loading in macrophages prolongs. Acetylated LDL in the cells decreased quickly and disappeared after 4 h of chasing. Subcellular fractionation using sucrose density gradient ultracentrifugation of macrophages, which had already accumulated OxPC-apoB complexes by 24 h of incubation with OxLDL and further 24 h chasing, showed that the complex was co-localized with endosomal and lysosomal markers. Immunohistochemical double staining studies demonstrated that OxPC and apoB co-localize in foam cells in early atherosclerotic lesions obtained from human coronary artery. These results suggest that OxPC-apoB complexes originating from OxLDL accumulate in foam cells in human atherosclerotic lesions as well as in macrophages in vitro.

Animals↗
PubMed1999

Taxonomic hierarchy of HLA class I allele sequences.

The markedly high levels of polymorphism present in classical class I loci of the human major histocompatibility complex have been implicated in infectious and immune disease recognition. The large numbers of alleles present at these loci have, however, limited efforts to verify associations between individual alleles and specific diseases. As an approach to reduce allele diversity to hierarchical evolutionarily related groups, we performed phylogenetic analyses of available HLA-A, B and C allele complete sequences (n = 216 alleles) using different approaches (maximum parsimony, distance-based minimum evolution and maximum likelihood). Full nucleotide and amino acid sequences were considered as well as abridged sequences from the hypervariable peptide binding region, known to interact in vivo, with HLA presented foreign peptide. The consensus analyses revealed robust clusters of 36 HLA-C alleles concordant for full and PBR sequence analyses. HLA-A alleles (n = 60) assorted into 12 groups based on full nucleotide and amino acid sequence which with few exceptions recapitulated serological groupings, however the patterns were largely discordant with clusters prescribed by PBR sequences. HLA-B which has the most alleles (n = 120) and which unlike HLA-A and -C is thought to be subject to frequent recombinational exchange, showed limited phylogenetic structure consistent with recent selection driven retention of maximum heterozygosity and population diversity. Those allele categories recognized offer an explicit phylogenetic criterion for grouping alleles potentially relevant for epidemiologic associations, for inferring the origin of MHC genome organization, and for comparing functional constraints in peptide presentation of HLA alleles.

Alleles↗
PubMed1976

T-lymphocyte receptors for alloantigens.

Recent developments in research on T-cell receptors for alloantigens have been reviewed. Recognition of these antigens is T-cell dependent and, when measured in the PAR test, can be shown to take place with receptors present on T cells, with those shed spontaneously from T cells and also with "recognition structures" of as yet unknown origin and structure present in post-transplantation alloantiserum but absent from B-cell induce alloantiserum. All recognizing structures of parent T-cell origin mentioned above also induce formation of anti-T cell receptor antisera when injected into appropriate F1 hybrid animals. The highly specific inhibitory activity of the sera can be demonstrated both in vitro and in vivo, and the most fascinating aspect constitutes cytotoxic elimination of T cells bearing receptors for a given alloantigenic specificity without harming T cells with other alloreceptors. The biochemical nature of T-cell receptors is not known and, depending on the physical form in which it is investigated, high molecular weight as well as considerably lower molecular weight structures have been found. It has, on the other hand, become quite clear that the antigen-binding region of B- and T-cell receptors shows idiotypic similarity and it is, therefore, likely that this part of the receptor molecule of both lymphocyte classes is similar if not identical

Animals↗
PubMed2002

Purification of two types of gonadotropin receptors from carp ovarian follicles: overlapping recognition by two different ligands.

Teleostean gonadotropin receptors were solubilized from the plasma membrane preparation of carp ovarian follicles by lithium diiodosalicylate and Triton X-100. Solubilization resulted fourfold increase in GTH binding activity as compared to the crude plasma membrane preparation. An addition of 25% glycerol and protease inhibitors to the solubilized receptor retained more than 90% original activity at -20 degrees C for 30 days. Gel filtration through Sephadex G 100 significantly increased the specific binding capacity from 70fmol/mg protein (soluble receptor) to 250fmol/mg protein. Peak I of gel filtration showing the receptor activity was further purified by affinity chromatography on purified salmon GTH II - Sepharose with a remarkable increase in specific binding capacity from 250fmol/mg protein (gel filtration peak) to 2300pmol/mg protein for salmon GTH I ligand and 2800pmol/mg protein for GTH II ligand. In SDS-PAGE affinity eluate the active peak showed two distinct bands corresponding to 66 and 62kDa molecular masses. These two proteins were clearly separated in FPLC Mono S chromatography, 62kDa as GTHR I and 66kDa as GTHR II. The former preferably binds to GTH I ligand, while the latter to GTH II, although both demonstrated overlapping recognition to both the ligands. GTH receptor protein I (GTHR I) and II (GTHR II) were purified 42,000- and 54,000-fold, respectively. Competitive binding inhibition studies indicate GTH I and ovine FSH a better ligand for GTHR I, while GTH II and ovine LH were preferable ligands for GTHR II. Biological relevance of these two receptor proteins was ascertained by monitoring the specific binding capacity of GTHR I and II at different stages of the annual reproductive cycle. GTHR I-GTH I was a prevailing complex during preparatory and pre-spawning stages, while GTHR II-GTH II was the dominant one at the maturational and final maturational stages. It may be concluded there are two GTH receptor proteins, each having a preferred ligand. The overlapping ligand-binding activity and profile of each receptor ligand complex suggest their link to seasonal development and maturation of carp ovarian follicle.

Animals↗
PubMed2001

Kinetic characterization of the pH-dependent oligomerization of R67 dihydrofolate reductase.

Protein-protein recognition results from the assembly of complementary surfaces on two molecules that form a stable, noncovalent, specific complex. Our interest was to describe kinetic aspects of the recognition in order to understand the subtle molecular mechanism of association. R67 dihydrofolate reductase (DHFR) provides an ideal model to investigate kinetic parameters of protein-protein association since it is a homotetramer resulting from the pH-dependent dimerization of homodimers. We took advantage of the presence of a tryptophan residue at the dimer-dimer interface to monitor pH-dependent oligomerization of R67 DHFR using stopped-flow fluorescence techniques. Except for pH near neutrality where dissociation exhibited biphasic kinetics, association and dissociation followed monophasic kinetics fitted on a two-state model. Apparent rate constants of association k(on) and dissociation k(off) were determined at various pHs and pointed to the key role of a histidine located at the dimer-dimer interface in the pH control of tetramerization. The values of the tetramer-dimer equilibrium dissociation constant were calculated from the ratio k(off) /k(on) and correlated well with those previously measured at equilibrium. The thermodynamic parameters and the activation energies of both the association and dissociation were determined and indicated that the association is enthalpy driven and suggested that the formation of four hydrogen bonds (one per monomer) is responsible for the thermodynamic stability of the tetramer. Detailed analysis of the biphasic kinetics led to an original model, in which protonation of the tetramer is the triggering event for the dissociation process while the association involves primarily the unprotonated dimers.

Dimerization↗
PubMed2006

Costimulatory receptors in a teleost fish: typical CD28, elusive CTLA4.

T cell activation requires both specific recognition of the peptide-MHC complex by the TCR and additional signals delivered by costimulatory receptors. We have identified rainbow trout sequences similar to CD28 (rbtCD28) and CTLA4 (rbtCTLA4). rbtCD28 and rbtCTLA4 are composed of an extracellular Ig-superfamily V domain, a transmembrane region, and a cytoplasmic tail. The presence of a conserved ligand binding site within the V domain of both molecules suggests that these receptors likely recognize the fish homologues of the B7 family. The mRNA expression pattern of rbtCD28 and rbtCTLA4 in naive trout is reminiscent to that reported in humans and mice, because rbtCTLA4 expression within trout leukocytes was quickly up-regulated following PHA stimulation and virus infection. The cytoplasmic tail of rbtCD28 possesses a typical motif that is conserved in mammalian costimulatory receptors for signaling purposes. A chimeric receptor made of the extracellular domain of human CD28 fused to the cytoplasmic tail of rbtCD28 promoted TCR-induced IL-2 production in a human T cell line, indicating that rbtCD28 is indeed a positive costimulator. The cytoplasmic tail of rbtCTLA4 lacked obvious signaling motifs and accordingly failed to signal when fused to the huCD28 extracellular domain. Interestingly, rbtCTLA4 and rbtCD28 are not positioned on the same chromosome and thus do not belong to a unique costimulatory cluster as in mammals. Finally, our results raise questions about the origin and evolution of positive and negative costimulation in vertebrate immune systems.

Amino Acid Sequence↗
PubMed1996

Amblyopic quasi-blindness for image structure.

Human amblyopes display reduced contrast sensitivities, suffer from perceptual distortion, and their letter acuities are worse than is predicted from grating visibility. We sought the origin of these dysfunctions by measuring normal and amblyopic sensitivities to various forms of well-defined image distortion, namely band-limited phase quantization, phase quantization with additional amplitude modulation, and grey-scale modification. Our results prove the existence of an amblyopic quasi-blindness to image structure, that cannot be explained in terms of contrast detection. We discuss these findings within the computational scheme of image decomposition into local amplitude and local phase values. they are consistent with the assumption of amblyopic eyes beings impaired in processing local phase but having the local amplitude (or "energy", possibly at reduced gain) at their disposal. Phrased in physiological terms, we propose a scheme of complex-cells-only vision in amblyopia. We also provide a demonstration of how amblyopic eyes may see the test stimuli and natural images by generating local amplitude and phase representations at limited phase resolution.

Amblyopia↗
PubMed1991

Tandem mass spectrometry identifies sites of three post-translational modifications of spinach light-harvesting chlorophyll protein II. Proteolytic cleavage, acetylation, and phosphorylation.

The photosynthetic membranes of spinach (Spinacia oleracea L.) chloroplasts were incubated with [gamma-32P] ATP. When the thylakoid membrane kinase was activated with light, the 25- and 27-kDa forms of the light-harvesting chlorophyll a/b protein (LHC II) were phosphorylated on their amino termini. Treatment of the membranes with proteinase K or thermolysin released phosphopeptides which were purified by ferric ion affinity chromatography and reverse phase high performance liquid chromatography. Sequencing of the phosphopeptides was performed with tandem quadrupole mass spectrometry. Three different phosphopeptides Ac-RKTAGKPKT, Ac-RKTAGKPKN, and Ac-RKSAGKPKN originating from class I LHC II were examined after release by thermolysin. One phosphopeptide, Ac-RRTVKSAPQ, originating from class II LHC II was examined after release by proteinase K. Each of the four LHC II phosphopeptides was derived from the amino terminus of a distinct protein. Peptides were acetylated at their amino-terminal arginine and were phosphorylated on either threonine or serine in the third position. We conclude that proteolytic processing of pre-LHC II occurs at a conserved methionyl-arginyl bond and is followed by amino-terminal acetylation of the arginine and nearby phosphorylation of the mature LHC II. Eight different peptides were synthesized in acetylated and nonacetylated forms as substrates for the thylakoid membrane kinase. From a comparison of the kinetics of phosphate incorporation into the peptides, we conclude that basic residues on both sides of the phosphorylation site are important for enzyme recognition. Acetylation of the amino terminus is not required for phosphorylation.

Acetylation↗
PubMed1997

Two domains of the AL1 protein mediate geminivirus origin recognition.

The geminiviruses tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) have bipartite genomes. Their A and B DNA components contain cis-acting sequences that function as origins of replication, while their A components encode the trans-acting replication proteins--AL1 and AL3. Earlier experiments demonstrated that virus-specific interactions between the cis- and trans-acting functions are required for TGMV and BGMV replication and that the AL1 proteins of the two viruses specifically bind their respective origins. In the current study, characterization of AL1 and AL3 proteins produced from plant expression cassettes in transient replication assays revealed that interaction between AL1 and the origin is responsible for virus-specific replication. The AL3 protein does not contribute to specificity but can be preferred by its cognate AL1 protein when replication is impaired. Analysis of chimeric proteins showed that two regions of AL1 act as specificity determinants during replication. The first domain is located between amino acids 1 and 116 and recognizes the AL1 origin binding site. The second region, which is between amino acids 121 and 209, is not dependent on the known AL1 DNA binding site. Analysis of wild type and chimeric proteins in transient transcription assays showed that AL1 also represses its own promoter in a virus-specific manner. Transcriptional specificity is conferred primarily by AL1 amino acids 1-93 with amino acids 121-209 making a smaller contribution. Together, these results demonstrated that the virus-specific interactions of AL1 during replication and transcription are complex, involving at least two discreet domains of the protein.

Amino Acid Sequence↗