Site-specific mutagenesis of potato spindle tuber viroid cDNA:

Summary The infectivity of cloned viroid cDNAs permits investigation of structure/function relationships in these unusual pathogenic RNAs by systematic site-specific mutagenesis of the cDNAs and subsequent bioassay. We have used three different strategies to create nucleotide substitutions within premelting region 2, a region of potato spindle tuber viroid (PSTV) believed to be important in viroid replication: sodium bisulfitecatalyzed deamination of deoxycytosine residues, oligonucleotide-directed mutagenesis, and construction of chimeric viroid cDNAs from fragments of infectious PSTV and tomato apical stunt viroid cDNAs. Although their effects upon the rod-like native structure of PSTV should be minimal, C U transitions at positions 92 or 284 appeared to be lethal. When inoculation with PSTV cDNA containing a single nucleotide substitution was mediated by the Ti plasmid of Agrobacterium tumefaciens, PSTV progeny with an unaltered wild type sequence was obtained. Two factors, the high error frequency characteristic of RNA synthesis and the use of a systemic bioassay for PSTV replication, may explain such sequence reversion and emphasize the importance of an appropriate bioassay system for screening mutant viroid cDNAs.     

The α/β Interfaces of α1β1, α3β3, and F1: Domain Motions and Elastic Energy Stored during γ Rotation

ATP synthase (F_oF₁) consists of F₁ (ATP-driven motor) and F_o (H⁺-driven motor). F₁ is a complex of ₃₃ subunits, and is the rotating cam in ₃₃. Thermophilic F₁ (TF₁) is exceptional in that it can be crystallized as a monomer and an ₃₃ oligomer, and it is sufficiently stable to allow refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified or . The nucleotide-dependent open–close conversion of conformation is an inherent property of an isolated and energy and signals are transferred through / interfaces. The catalytic and noncatalytic interfaces of both mitochondrial F₁ (MF₁) and TF₁ were analyzed by an atom search within the limits of 0.40 nm across the interfaces. Seven (plus thermophilic loop in TF₁) contact areas are located at both the catalytic and noncatalytic interfaces on the open form. The number of contact areas on closed increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces. The interfaces in the barrel domain are immobile. The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in . The notion of elastic energy in F_oF₁ has been revised. X-ray crystallography of F₁ is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of F_oF₁. The domain motion and elastic energy in F_oF₁ will be elucidated by time-resolved crystallography.     

Synthesis of chemically modified sialic acid-containing sialyl-LeX ganglioside analogues recognized by the selectin family

Sialyl Lewis X ganglioside analogues containing 5-acetamido-3,5-dideoxy-l-arabino-2-heptulopyranosylonic acid (C7-Neu5Ac), 5-acetamido-3,5-dideoxy-d-galacto-2-octulopyranosylonic acid (C8-Neu5Ac), and 5-acetamido-3,5-dideoxy-l-glycero-d-galacto-1-2-nonulopyranosylonic acid (8-epi-Neu5Ac) in place ofN-acetylneuraminic acid (Neu5Ac) have been synthesized. Glycosylation of 2-(trimethylsilyl)ethyl 6-O-benzoyl--d-galactopyranoside with the phenyl or methyl 2-thioglycoside derivatives of the respective sialic acids, usingN-iodosuccinimide (NIS)-trifluoromethanesulfonic acid as a promoter in acetonitrile, gave the three required 2-(trimethylsilyl)ethyl (2S)-sialyl-(2 3)--galactopyranosides. These were converted viaO-benzoylation, selective transformation of the 2-(trimethylsilyl)ethyl group to acetyl, and introduction of the methylthio group with methylthiotrimethylsilane into the corresponding glycosyl donors. Glycosylation of 2-(trimethylsilyl)ethylO-(2,3,4-tri-O-benzyl--l-fucopyranosyl)-(1 3)-O-(2-acetamido-6-O-benzyl-2-deoxy--d-glucopyranosyl)-(1 3)-2,4,6-tri-O-benzyl--d-galactopyranoside with these donors in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) afforded the expected -glycosides, which were converted into the corresponding -trichloroacetimidates, and these, on coupling with (2S, 3R, 4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol, gave the required -glycosides. Finally, these were transformed via selective reduction of the azide group, condensation with octadecanoic acid,O-deacylation, and de-esterification into the target compounds in good yields.     

Allelic relationships between genes for resistance to tomato spotted wilt tospovirus in Capsicum chinense

Pepper (Capsicum chinense Jacq.) has been reported to be an important reservoir of resistance genes to tomato spotted wilt virus (TSWV). The genes for TSWV resistance present in three C. chinense lines (PI 152225, PI 159236 and Panca) were investigated for allelism. All resistant lines were crossed with each other. Parents, F₁, backcrosses and F₂ populations (including reciprocals) developed from those crosses were mechanically inoculated with a highly virulent TSWV isolate. Susceptible C. annuum cv Magda was used to check inoculum virulence. Fifty plants of the F₁ hybrids; Magda x PI 152225, Magda x PI 159236, and Magda x 'Panca, were also inoculated with the TSWV isolate. The resistance response in all C. chinense sources was associated with a localized, hypersensitive-like reaction that was phenotypically expressed as a prompt formation of large local lesions accompanied by premature leaf abscission. All F₁ generations presented a final score of resistant; indicating that the expression of resistance to TSWV is conditioned by a dominant gene regardless of the source. The absence of segregation for resistance to TSWV that was observed in all generations of the crosses between C. chinense lines indicated that either a tightly linked group of genes exists or that the resistance is governed by the same single major gene (probably the already described Tsw gene). Previous reports have indicated that the Tsw gene is not effective against tospovirus members of serogroup II, i.e. tomato chlorotic spot virus (TCSV) and groundnut ring spot virus (GRSV). In the assay described here, all of the C. chinense lines showed, after mechanical inoculation, an identical susceptibility response to the TCSV and GRSV isolates.     

Brain Net Unidirectional Uptake of α-[14C]Methyl-L-Tryptophan (α-MTrp) and Its Correlation with Regional Serotonin Synthesis,Tryptophan Incorporation into Proteins,and Permeability Surface Area Products of Tryptophan and α-MTrp

The uptake and trapping constants for labeled tryptophan (Trp) via the serotonin (5-hydroxytryptamine; 5-HT) metabolic pathway and for the incorporation of Trp into proteins, and -[¹⁴C]methyl-L-tryptophan (-MTrp) were measured. Measurements were done in rats treated with either saline or probenecid (200 mg/kg). In addition, the blood-brain barrier (BBB) permeability surface area products for Trp (PS_T) and -MTrp (PS) were measured in normal rats. The results suggest that, in both groups of rats, there is a highly significant correlation (p < 0.05; Pearson Product Moment Correlation (PPMC) between the brain uptake and trapping constants for -MTrp and those of Trp via the 5-HT metabolic pathway, but there is no significant correlation (p > 0.05; PPMC) between either of these constants and the PS products of either compound. There is also no significant correlation (p > 0.05; PPMC) between the constant for the Trp incorporation into proteins with any of the other parameters. For all parameters, except Trp incorporation into proteins (-MTrp is not incorporated into proteins), there was a highly significant correlation (p < 0.001) between the quantities measured for Trp and -MTrp. The data presented here strongly suggests that the brain uptake and trapping of -MTrp relates to brain 5-HT synthesis, and does not relate to the BBB transport or protein incorporation of Trp. On the basis of these results, as well as those previously reported, we concluded that trapping (unidirectional uptake) of -MTrp can be converted to the 5-HT synthesis rates in the brain. From this also follows that labeled -MTrp is a good tracer for in vivo evaluation of the brain 5-HT synthesis.     

The systematics of Porphyra: character evolution in closely related species

Isozymes, vegetative and reproductive morphology, seasonality, vertical and geographic distributions and chromosomes were compared for six pairs of putative sibling species of Porphyra (P. abbottae/P. torta, P. fallax subsp. fallax/P. fallax subsp. conwayae, P. amplissima/P. cuneiformis, P. fucicola/P. leucostica, P. miniata/P. variegata, P. umbilicalis/P. umbilicalis) and among five species in a complex (P. brumalis, P. kurogii, P. linearis, P. pseudolinearis, and P. purpurea.) Geographic distribution and zymograms for certain proteins showed the greatest change between species pairs: only one pair of species had identical distributions, and most species pairs were disjunct; every species had a different allozyme for GOT-1, whereas all species had apparently identical proteins for phycoerythrin. Seasonality and habitat exhibited moderate differentiation: Northeast Pacific sibling species were characterized by a high intertidal winter species pairing with a mid intertidal spring species, whereas all but one of the other species pairs exhibited nearly identical vertical distributions and seasonalities. There were few changes in morphology: most species pairs had essentially identical morphologies and coloration and the same arrangement of reproductive cells. Chromosome numbers and karyotypes were identical for species pairs and in the species complex. These results provide evidence for different rates of evolution of different characters in the genus Porphyra.     

Asymmetric hybridization between cytoplasmic male-sterile (CMS) and fertile rice (Oryza safiva L.) protoplasts

Summary ⁶⁰Co-irradiated protoplasts of the cytoplasmic male-sterile line A-58 CMS (Oryza saliva L.) were electrofused with iodoacetamide (IOA)-treated protoplasts of the fertile (normal) rice cultivar Fujiminori. Seven of the colonies that formed were identified as cytoplasmic hybrids (cybrids): they all had the peroxidase isozymes of the fertile Fujiminori parent, but contained four plasmid-like DNAs (Bl, B2, B3 and B4) from the sterile A-58 CMS parent in their mitochondrial genomes. In addition, digestion of cybrid mtDNA gave a set of restriction fragments that differed from those of the parents.     

Immortalized human endothelial cells have a decreased response to IL-1 secondary to an IL-1 receptor defective expression restored by corticosteroids

A human endothelial cell line is a convenient tool for exploring cell physiology and testing drugs and toxics. Several attempts have been made using SV40 to immortalize endothelial cells. We used human umbilical vein endothelial cells (HUVEC) transformed with a construct made of promoter of the vimentin gene and SV40 Tag. The proliferation of immortalized vascular endothelial cells (IVEC), as measured by [methyl-³H]thymidine incorporation, was compared to that of HUVEC in the presence of endothelial cell growth factor and cytokines: tumor necrosis factor- (TNF-), interleukin-1 (IL-1) and interferon- (IFN-). Inhibition of [methyl-³H]thymidine incorporation by IL-1 was lower than that observed with HUVEC, while TNF- reduced the proliferation of IVEC and HUVEC to similar extents. Induction of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin by TNF-, measured by a radiometric technique, was similar in IVEC and HUVEC, while the induction of E-selectin by IL-1 on IVEC was limited and significantly different from that observed on HUVEC (p<0.001). The number of ¹²⁵I-IL-1 binding sites on IVEC is 3-fold less than on HUVEC and the IL-1 receptor number was reduced. Dexamethasone treatment of IVEC restored their reactivity to IL-1 and corrected the IL-1 binding and the receptor number. These results showed that the introduction of SV40 gene not only immortalized the cell but also altered IL-1 receptor expression. This alteration may be improved by addition of corticosteroids to the cell culture, which extends the possibility of using IVEC as a model of endothelial cells.     

Immunohistochemical localization of serine-protease inhibitors in the human placenta

Proteases and their inhibitors play a pivotal role in developmental and differentiative processes. In the present report we investigated the immunohistochemical localization of 1-antitrypsin, 1-antichymotrypsin and inter--trypsin inhibitor in first trimester as well as in term human placentas. For this purpose polyclonal antibodies against these serine-protease inhibitors were used. All inhibitors were expressed in the villous syncytiotrophoblast of first and last trimester placentas. Placental fibrinoid was positively stained for 1-antitrypsin and inter--trypsin inhibitor throughout gestation. 1-Antitrypsin and 1-antichymotrypsin showed a strong immunostaining in the Hofbauer cells (first trimester and full term placentas). Extravillous cytotrophoblast was negative for the three protease inhibitors throughout gestation. The presence of the three inhibitors in the syncytiotrophoblast suggests a role in coagulative, invasive and immunomodulatory processes. Fibrinoid, staining for 1-antitrypsin and inter--trypsin inhibitor, could also have an important immunoprotective function. The presence of protease inhibitors in the Hofbauer cells suggests an involvement of these cells in villous remodelling and differentiative processes.     

Genes encoding acidic and basic class III β-1,3-glucanases are expressed in tomato plants upon viroid infection

-1,3-glucanases are hydrolytic enzymes considered to constitute part of the general array of defense genes induced by pathogen infection in higher plants. We have isolated and characterized two complementary DNA clones, corresponding to new -1,3-glucanases from tomato plants (Lycopersicon esculentum) which are expressed upon challenge with citrus exocortis viroid. Amino acid sequence comparison revealed that they are most similar to -1,3-glucanases from tobacco, particularly to PR-Q, the unique component of the class III -1,3-glucanase. The deduced amino acid sequences of the two tomato -1,3-glucanases indicate that, although being highly similar in amino acid sequence, they have different isoelectric points: pI 10.5 for the basic isoform (Tom PR-Q b) and pI 5.2 for the acidic one (Tom PR-Q a). The expression of these two -1,3-glucanase messenger RNAs (mRNAs) in response to viroid infection and ethephon treatments was examined. mRNAs for these two isoforms are coordinately expressed and induced similarly to mRNAs for other PR proteins, indicating that they are part of a general and coordinate mechanism of response of tomato plants susceptible to viroid infection.     

Identification and isolation of a unique esterase from the medium of non-embryogenic cell line of cultured carrot cells

A unique esterase isozyme z with very low electrophoretic mobility on the anionic polyacrylamide gel (PAGE) was found in the medium of a non-embryogenic (Ca-4) line of cultured carrot (Daucus carota L.) cells. The protein corresponding to this esterase isozyme z was purified by electroelution from preparative PAGE and the esterase migrated as a single band with an apparent M _r of 35 000 on SDS-PAGE. The purified esterase isozyme z exhibited at least 350-fold higher specific activity than that in the total medium proteins.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

In vitro regeneration of evergreen azalea from leaves

Rhododendron simsii Hellmut Vogel was regenerated using different types of explants, auxins and cytokinins. After a callus induction phase, with 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid, adventitious shoot regeneration was obtained on a medium supplemented with thidiazuron or zeatin. With thidiazuron shoots were small and a subsequent elongation step was required before rooting. An elongation step was not required when zeatin was used. The duration of the callus induction phase was negatively correlated with the regeneration capacity.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - 2iP 6-(--dimethylallylamino)purine - NOA ß-naphthoxyacetic acid - BA 6-benzyladenine - IBA 1-H-indole-3-butyric acid - IAA 1-H-indole-3-acetic acid - TDZ thidiazuron - WPM woody plant medium     

Characterization of the guanosine 3′∶5′-monophosphate-dependent protein kinase from silkworm eggs and analysis of the endogenous protein substrates

Summary In a previous paper, two types of cyclic nucleotide-dependent protein kinases, namely cGMP dependent G-kinase and cAMP dependent A-kinase, in silkworm eggs has been reported (Takahashi et al. 1975; Takahashi 1976). One of these, G-kinase, has now been purified 2400-fold by means of ammonium sulfate fractionation, chromatography on hydroxylapatite, DEAE cellulose, and gel filtration.Some of the properties of the enzyme are described. The enzyme is highly dependent on cGMP; it is strongly inhibited by GTP in a noncompetitive manner not only for ATP but also for cGMP. GTP was found to be highly inhibitory on G-kinases from various tissues of the silkworm, but did not inhibit the A-kinase.Incubation of the egg extract with [-³²P]ATP and Mg²⁺ led to the formation of three major³²P-labelled proteins, with molecular weights of 42.000, 70.000 and 180.000 as analyzed by SDS polyacrylamide gel electrophoresis. Two of them corresponded to the subunits of vitellin.The silkworm vitellin was effectively phosphorylated both by the highly purified G-kinase and by the A-kinase. It is concluded that the G-kinase is involved in the phosphorylation of vitellin in developing silkworm eggs.Abbreviations cAMP adenosine 35-monophosphate - cGMP guanosine 35-monophosphate - A-kinase adenosine 35-monophosphate-dependent protein kinase - G-kinase guanosine 35-monophosphate-dependent protein kinase - MIX 1-methyl-3-isobutylxanthine     

NMR structure of the ribosomal protein L23 from Thermus thermophilus

The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel -strands and three -helices connected as ------, and a large and flexible loop inserted between the third and fourth -strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.     

The polypeptide structure and assembly of Ly-2/3 heterodimers

Mild reduction of mature, thymic Ly-2/3 heterodimers of M _r 67 000 resulted in dissociation into three individual polypeptide chains, , , and , of respective M _r values 38000, 35000, and 30000. The and chains were both immunoprecipitated by a monoclonal antibody directed to the Ly-2.1 epitope whereas the Ly-3.1 antibody bound only the chain. The possibility that the and chains of each heterodimer established their interchain links within a labile precursor protein in which a and segments were fused was considered but discounted by the finding that in mice heterozygous for both Ly-2 and Ly-3 loci, the Ly-2 product of one chromosome was not exclusively joined to Ly-3 structures coded by the same chromosome. By utilizing ionic detergents which selectively alter the charge of intrinsic membrane proteins, both Ly-2 and Ly-3 polypeptides were shown to have membrane insertion sites. It is suggested that as a consequence of their likely synthesis on membrane-bound polysomes, newly synthesized Ly-2 and Ly-3 structures accumulate within the same subcellular compartment — the membranes of the rough endoplasmic reticulum. Their elevated concentration within this space may facilitate a low affinity binding interaction between Ly-2 and Ly-3 which is later stabilized by interchain disulfide bond formation.Abbreviations used in this paper BSA bovine serum albumin - DOC sodium deoxycholate - DTT dithiothreitol - HA hemagglutinin - HTAB hexadecyltrimethylammonium bromide - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TX100 Triton X-100     

E infα supk transgene in B10 mice suppresses the development of myasthenia gravis

Mice bearing the H-2 ^bhaplotype are susceptible to the development of experimental autoimmune myasthenia gravis (EAMG), induced by acetylcholine receptor (AChR) autoimmunity. One of the genes influencing EAMG susceptibility has been mapped to the A ^blocus of the major histocompatibility complex, and the A chain has been implicated in the pathogenesis. Mice of the H-2 ^bhaplotype, including C57BL/10 (B10), have a genomic deletion of the E gene and therefore fail to express the E molecule on their cell surface. To test the hypothesis that failure to express the cell surface E molecule in B10 mice contributes to EAMG pathogenesis, E _inf ^supk transgenic B10 mice expressing the T molecule were examined. Expression of the E molecule in E _inf ^supk transgenic B10 mice partially prevented the development of EAMG.     

Characterization of α-galactosidase isoenzymes in normal and fabry human-Chinese hamster somatic cell hybrids

Summary The -galactosidases in normal man-Chinese hamster somatic cell hybrids were investigated with antibodies specific for human -galactosidase A and antibodies specific for Chinese hamster -galactosidase. It was found that an isoenzyme in hybrid cells, which has an electrophoretic mobility between that of human -galactosidase A and Chinese hamster -galactosidase, contains immunologic determinants of both human and Chinese hamster origin, suggesting that it is a heteropolymeric molecule. Moreover, the locus for human -galactosidase, which was found to be X-linked, is the locus coding for -galactosidase A. Hybrids isolated after fusion of Chinese hamster cells with cells of a patient with Fabry's disease did not express human -galactosidase A or the heteropolymeric molecule even in the presence of the active human X chromosome, indicating that the deficiency of -galactosidase A in Fabry's disease is probably due to a mutation in a structural gene resulting in the inability to form immunologically detectable and functionally active molecules of -galactosidase A.     

Chromosome alterations in the karyotype of triticale in comparison with the parental forms

Summary R genome chromosomes were studied in two forms of primary triticales (hexaploid TPG-1/1-78 and octoploid AD 825) and in their parent rye forms (Secale cereale L. cv. Kharkovskaya 60 and VSKhI, respectively) using the methods of C-banding and morphometric analysis. The size of some heterochromatic segments was shown to alter in the karyotype of triticale. An increase in size was detected approximately in half of all telomeric C-bands; the size of the other C-bands either decreased or did not change. The frequencies of these alterations were 11. The variability in the size of telomeric C-bands in rye chromosomes diminished in both triticales studied. The two triticale forms inherited variants of R genome chromosome polymorphism predominantly with the medium size range of telomeric C-bands. The centromeric C-bands in both triticale forms either enlarged or did not alter. Possible mechanisms responsible for the observed pattern of alterations are discussed.     

Genetic mapping of mutation in Escherichia coli leading to a temperature-sensitive RNase D

Summary In order to determine the metabolic role of RNase D in Escherichia coli, we have attempted to isolate strains deficient in this enzyme. One strain containing a temperature-sensitive RNase D was found among a heavily mutagenized stock of strains temperature-sensitive for growth. Genetic mapping of the mutation responsible for the altered RNase D enabled us to define the rnd locus, at 39.5–40.0 min on the E. coli map, which apparently specifies the RNase D structural gene. Using a Tn10 insertion near the rnd locus, we constructed isogenic strains containing RNase D and RNase II mutations, alone or in combination. Although the original mutant isolate displayed temperature-sensitive growth, no growth phenotype was associated with the rnd mutation in wild type background, possibly because a substantial amount of RNase D remained in cells grown at 45° C. However, elucidation of the map position of the rnd locus should prove useful for the isolation of other mutant strains with lower levels of RNase D.This is paper 34 in the series Reactions at the 3 Terminus of tRNA. The previous paper in this series is Cudny et al. (1981 c)     

Bioconversion of elaidic acid to Δ9-trans-1,18-octadecenedioic acid by Candida tropicalis

Summary The bioconversion of elaidic acid to ⁹ acid by the mutant S₇₆ of Candida tropicalis was studied. It was found that the mutant could convert elaidic acid to ⁹ acid through the -oxidation of the terminal methyl group. The molecular formula and geometric configuration of the product were determined by different analytical methods in the absence of an authentic standard. The results demonstrated that no change in the geometric configuration occured during the conversion of the unsaturated monoic acid to ,-dioic acid. The production of this dioic acid reached 10 mg/ml in medium for 36 h with a conversion rate of 40%. Degradation pathways of this dioic acid are basically similar to the dioic acid derived from oleic acid by the same strain except for a small amount of heptanedioic acid which was observed in the medium. The IR spectrum, mass-spectrum, ¹H-NMR and ¹³C-satellite spectra of this compound are presented.