The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens

VirB9 and VirB7 are essential components of the putative VirB membrane channel required for transfer of the T-complex from Agrobacterium tumefaciens into plants. In this report, we present a biochemical analysis of their interaction and cellular localization. A comparison of relative electrophoretic mobilities under nonreducing and reducing conditions suggested that they form thiol-sensitive complexes with other proteins. Two-dimensional gel electrophoresis identified one complex as a heterodimer of VirB9 and VirB7 covalently linked by a disulfide bond, as well as VirB7 homodimers and monomers. Immunoprecipitation with VirB9-specific antiserum isolated the heterodimeric VirB9-VirB7 complex. Incubation with reducing agent split the complex into its constituent VirB9 and VirB7, which further confirmed linkage via cysteine residues. The interaction between VirB9 and VirB7 also was observed in the yeast two-hybrid system. Membrane attachment of VirB9-VirB7 may be conferred by lipoprotein modification, since labeling with [3H]palmitic acid in A. tumefaciens verified that VirB7 is a lipoprotein associated with VirB9. VirB9 and VirB7 showed equal distribution between inner and outer membranes, in accord with their proposed association with the transmembrane VirB complex.     

Stability of the Agrobacterium tumefaciens VirB10 protein is modulated by growth temperature and periplasmic osmoadaption

Export of oncogenic T-DNA from the phytopathogen Agrobacterium tumefaciens is mediated by the products of the virB operon. It has recently been reported (K. J. Fullner and E. W. Nester, J. Bacteriol. 178:1498-1504, 1996) that DNA transfer does not occur at elevated temperatures; these observations correlate well with much earlier studies on the temperature sensitivity of crown gall tumor development on plants. In testing the hypothesis that this loss of DNA movement reflects a defect in assembly or maintenance of a stable DNA transfer machinery at high temperature, we have found that steady-state levels of VirB10 are sensitive to growth temperature while levels of several other VirB proteins are considerably less affected. This temperature-dependent failure to accumulate VirB10 is exacerbated in an attachment-deficient mutant strain (chvB) which exhibits pleiotropic defects in periplasmic osmoadaption, and virulence of a chvB mutant can be partially restored by lowering the temperature at which the bacteria and the plant tissue are cocultivated. Furthermore, the stability of VirB10 is diminished in cells lacking functional VirB9, but only under conditions of low osmolarity. We propose that newly synthesized VirB10 is inherently labile in the presence of a large osmotic gradient across the inner membrane and is rapidly degraded unless it is stabilized by VirB9-dependent assembly into oligomeric complexes. The possibility that VirB10-containing complexes are not assembled properly at elevated temperatures suggests an explanation for the decades-old observation that tumor formation is exquisitely sensitive to ambient temperature.     

An isoflavonoid-inducible efflux pump in Agrobacterium tumefaciens is involved in competitive colonization of roots

Agrobacterium tumefaciens 1D1609, which was originally isolated from alfalfa (Medicago sativa L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn5-B30. Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame, ifeA, of a three-gene locus (ifeA, ifeB, and ifeR), which shows high homology to bacterial efflux pump operons. Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (P < or = 0.01) in competition against wild-type strain 1D1609. Site-directed mutagenesis experiments, which produced strains I-4 (ifeA::gusA) and I-6 (ifeA::omega-Tc), confirmed the importance of ifeA for competitive root colonization. Exposure to the isoflavonoid coumestrol increased beta-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined. In the same test, coumestrol accumulation in mutant strain I-6 did not decline. Expression of the ifeA-gusA reporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa. These results show that an efflux pump can confer measurable ecological benefits on A. tumefaciens in an environment where the inducing molecules are known to be present.     

Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called omega that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the omega region are not essential for nuclear uptake of T-DNA, and further suggested that the omega domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the omega domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of beta-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid (X-gluc). However, using an omega-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 omega deletion/substitution. Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an omega mutation. In addition, a mutant VirD2 protein lacking the omega domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the omega and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the omega domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.     

Cloning and characterization of a tetracycline resistance determinant present in Agrobacterium tumefaciens C58

Agrobacterium tumefaciens C58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. We observed that a mutation affecting a tRNA processing function significantly affected the emergence of such mutants, suggesting that C58 contained a positively acting gene conferring resistance to tetracycline. A cosmid clone conferring resistance to tetracycline in Escherichia coli and Agrobacterium was isolated from a genomic bank of one such mutant. Subcloning, transposon mutagenesis, and DNA sequence analysis revealed that this DNA fragment contained two divergently transcribed genes, tetA and tetR, encoding products that were very similar to proteins of the Tet(A) class of tetracycline resistance systems. In the clone from this mutant, tetR was disrupted by an IS426. The homologous region from wild-type NT1 contained an intact tetR gene and did not confer resistance to tetracycline. Hybridization analysis showed that of 22 members of the genus Agrobacterium surveyed, only strains C58 and T37 contained the tet determinant. Moreover, only these two strains mutated to resistance to this antibiotic. Unlike other Tet(A) systems, neither tetracycline nor a series of its derivatives induced the expression of this tet gene unit. Other polycyclic compounds, including many of plant origin, also did not induce this tet gene system. The divergent promoter region of this tet system contained a single inverted repeat element identical to one such operator repeat in the promoter region of the tet determinant from the IncP1alpha R plasmid RP4. TetR repressor proteins from the Agrobacterium tet system and from RP4 interacted with the heterologous operators. While the repressive effect of the TetR protein from strain C58 (TetRC58) on the tetA gene from strain RP4 (tetARP4) was not relieved by tetracycline, repression of tetAC58 by TetRRP4 was lifted by this antibiotic.     

Interactions between transport of triiodothyronine and tryptophan in JAR cells

We studied the effect of a number of amino acids on uptake of L-triiodothyronine (T3) in the human choriocarcinoma cell line, JAR. Tryptophan inhibited saturable T3 uptake by about 57% without any significant effect on the non-saturable uptake. Michaelis constant (Km) for T3 uptake was 1.06 +/- 0.15 microM (n = 15) with the corresponding maximum velocity (Vmax) of 24.2 +/- 3.1 pmol/min/mg cellular protein. For tryptophan uptake the Km was 1.31 +/- 0.26 microM (n = 7) and Vmax was 166.4 +/- 35.7 pmol/min/mg protein. The kinetic parameters for both uptake processes were similar to those reported in normal placenta. Uptake of T3 was inhibited by tryptophan but not phenylalanine, but tryptophan uptake was inhibited both by T3 and phenylalanine. Inhibition of T3 uptake by tryptophan was dose dependent, with an inhibition constant (Ki) of 2.9 +/- 0.5 mM. Similarly, tryptophan uptake was inhibited by T3 and phenylalanine in a dose dependent way with Ki values of 4.9 +/- 0.5 microM and 15.6 +/- 4.8 microM respectively. Km for T3 uptake was significantly increased to 1.86 +/- 0.42 microM (n = 4) in the presence of 3 mM unlabelled tryptophan and, similarly, Km for tryptophan uptake was significantly increased to 9.91 +/- 2.57 microM (n = 3) in the presence of 5 microM unlabelled T3. Efflux of T3 was progressively inhibited by increasing concentrations of both ligands, i.e. was saturable. We conclude that there is mutual competitive inhibition between uptake systems for T3 and tryptophan in JAR cells, but the kinetic parameters of cross-inhibition of uptake by the substrates suggest that the carriers are distinct. T3 may be transported in JAR cells by at least two transport systems with differing substrate specificities. We also demonstrated the presence of a saturable membrane carrier mediating the efflux of T3 from the cells which was subject to trans-inhibition by T3 and tryptophan.     

Interactions between membrane potential and intracellular calcium concentration in vascular smooth muscle

The intracellular calcium concentration is a major determinant of vascular tone. In the steady state it is regulated mainly by membrane potential. At the same time, several mechanisms regulating the calcium concentration, including the membrane potential, are influenced by the intracellular calcium concentration itself. There are thus multiple possible positive and negative feedback loops involved in calcium regulation. This review gives a brief overview of the different mechanisms involved, including calcium-dependent ion channels, exchangers, and ATPases, and discusses their role in agonist-mediated responses, in relation primarily to studies on the portal vein and mesenteric small arteries.     

Cholesterol-independent targeting of Golgi membrane proteins in insect cells

Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.     

Formation of deletional derivatives of the Ti-plasmid pGV3850 in a conjugative transfer from Agrobacterium tumefaciens to Escherichia coli

The process of the transfer of the Ti-plasmid vector pGV3850 with the plasmid pBR322 inserted into the T-DNA region from Agrobacterium tumefaciens to a non-plasmid strain of Escherichia coli was studied. The transferred Ti-plasmid was found to be exposed to deletions and formed a wide range of derivatives with a size ranging from 3-4 kb to 50 kb, maintained in E. coli due to ColE1-replicon. The Ti-plasmid is also inserted into the chromosome of the recipient bacterium with at least a 100-fold lower frequency than the formation of deletional derivatives. It was shown that the induction of vir genes controlling the transfer of T-DNA into plants has no appreciable effect on the efficiency of obtaining transconjugates in mating with E. coli. The deletion of the genetic material of megaplasmids with the inserted functional site OriV ColE1, as a result of the conjugative transfer from cells of different bacteria to the cells of E. coli, was proposed for molecular cloning.     

Bean Dwarf mosaic geminivirus movement proteins recognize DNA in a form- and size-specific manner

Various ratios of succinic acid to fenoldopam mesylate, ranging from 0:1 to 18:1 were incorporated in pellets and coated with 1.5-12% w/w Surelease. Even though the coating level did influence the rate and amount of fenoldopam release, the influence of the succinic acid to drug ratio was much more important and evident at all coating levels. Being a weakly basic drug, fenoldopam release ceased when testing in SIF for succinic acid to drug ratios of 0:1-4:1, with the end of release being more abrupt for the 0:1 than for the 4:1 ratio. Only for a succinic acid to drug ratio of > or =5 was fenoldopam release constant for 6-8 h and independent of the pH-value of dissolution media. For a thin coat of about 2.5% w/w Surelease, those pellets showed an ideal controlled release behaviour with release rates of about 5-10%/h and a total release of almost 80% in 8 h. The dissolution profiles of Surelease coated pellets with high succinic acid to drug ratios (> or =5) and different coating levels, were evaluated for best fits to commonly used kinetic models. Sustained release mechanisms are discussed according to best fit models. The quantification of the pH-adjuster succinic acid, released from pellets with an acid to drug ratio of < or =1 showed, that despite their failure as a controlled release system for fenoldopam, the investigated coats could control the release of succinic acid effectively at optimized coating levels. For increasing succinic acid to drug ratios (< or =4) succinic acid was released at an ever more constant rate and release rates, though still faster than the release rates of fenoldopam, decreased steadily for increasing ratios. At a 5:1 ratio finally release rates of succinic acid and fenoldopam were almost identical. Therefore those pellet cores were almost completely emptied during dissolution testing, with both fenoldopam and succinic acid leaving at a constant rate and a total release of about 80% each for a 2.5% Surelease coat, while lower succinic acid to drug ratios had failed to show any sustained release for such thin Surelease coats. A similar formulation with fumaric acid instead of succinic acid failed to show the desired release pattern, indicating that it is the presence of a sufficiently high amount of succinic acid rather than the presence of an acidic compound in general, that ensures fenoldopam solubility at higher pH-values.     

Isoelectric focusing of plasma membrane proteins from Strongylocentrotus intermedius sea urchin embryo cells

14C-amino acids were introduced into the developing sea urchin embryos S. intermedius in 7,5 hours after the fertilization (middle blastula stage). In 30 min the embryos development were terminated, the plasma membrane was isolated and 14C-labelled proteins were recovered from the membrane with 8 M urea and separated by isoelectrofocusing in pH gradient 3,5-10 in presence of 6 M urea. All 14C-label was introduced into two proteins with pI 4,85-4,90 and 5,20-5,25 and the proteins were effectively separated. The minimal molecular weight of the proteins estimated by SDS-acrylamide gel-electrophoresis was 10 000 and 15 000 daltons, respectively.     

Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis

We report the clinicopathologic characteristics of pulmonary lymphomatoid granulomatosis (LYG) in 11 patients (identified from a series of 330 consecutive patients who underwent autopsy between 1984 and 1995 at the University of Texas Medical Branch at Galveston, Texas) with a diagnosis of acquired immunodeficiency syndrome (AIDS). We used immunohistochemical stains, RNA in situ hybridization (ISH), and gene rearrangement studies to identify the immunophenotype and the presence or absence of Epstein-Barr virus (EBV) infection. All of the patients were men ranging in age from 27 to 65 years (mean age, 38.6 yr). Autopsy lungs of 21 age-matched controls were examined for EBV using ISH; these included 9 patients with AIDS who did not have pulmonary lesions and 12 HIV-negative individuals who died accidentally (mean age, 38.6 yr). All of the 11 pulmonary lesions showed the gross and microscopic characteristics of LYG, with zonal necrosis and prominent angioinvasion. The tumor nodules consisted of a mixture of atypical large lymphocytes, with vesicular nuclei and prominent nucleoli and with a background of small and intermediate-size lymphocytes, histiocytes, and plasma cells. The large lymphocytes were CD20 positive, consistent with a B-cell phenotype. Ten of the 11 cases demonstrated EBV1-encoded RNA and CD20 positivity in the large, atypical lymphocytes by double labeling. One patient showed EBV positivity in CD20-negative, CD45RO-positive large cells, but these cells were CD3 negative and showed a monoclonal heavy chain gene rearrangement by polymerase chain reaction, indicating that these were of B-cell origin. Aberrant CD43 coexpression was identified in four cases. EBV latent membrane protein was demonstrated in 9 of 11 cases by immunohistochemical stains. The lungs of all of the 21 control patients were negative for EBV by ISH. We conclude that, in our series, AIDS-associated LYG is a B-cell neoplasm and that it has a strong association with EBV infection.     

Interactions between contact and chemosensory mechanisms in pain modulation in 10-day-old rats.

Antinociception was evaluated in 10-day-old rats while suckling or in contact with the mother. Testing occurred during, immediately after, or at 30 and 60 sec following milk-induced hyperextension. Hyperextension-induced hypoalgesia terminated immediately with stretch cessation. For suckling rats, baseline escape levels were reachieved within 1 min. For contact rats, baseline levels were also manifest at 30 sec but were elevated by 1 min. Sublingual infusions into the anterior portion of the mouth in rats that were either suckling or in contact caused a 20–25-sec increase in escape latencies. For suckling rats, escape latencies returned to baseline levels immediately at infusion termination. For contact rats, latencies continued to be elevated for at least 5 min postinfusion. Thus, 3 classes of mother–infant interactions, contact, suckling, and hyperextension during milk letdown, cause varying degrees of hypoalgesia in 10-day-old rats. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The amino acid sequence of a protein from wheat kernel closely related to proteins involved in the mechanisms of plant defence

The effects of systemic administration of muscle extract on normal muscle were studied. Male Wistar rats received intraperitoneal injections of normal and denervated muscle extract over 5 consecutive days. Soleus muscles were then submitted to histological, histochemical and quantitative-morphometric analysis. The group receiving denervated muscle extract showed considerable muscle fiber hypertrophy, together with the formation of new fibers suggestive of hyperplasia. The systemic administration of denervated muscle extract was shown to have a considerable myotrophic effect on normal muscle, evident in the hypertrophy and hyperplasia of muscle fibers.     

Purification and partial sequence of proteins involved in the cholic acid transport into rat liver hepatocytes

Two proteins, in previous work labeled by affinity markers derived from taurocholic acid, were purified and partially sequenced. Antibodies were raised against purified proteins, and cross-reactions were carefully checked. The influence of these antibodies upon taurocholic acid import into vesicles from rat liver plasma membranes was measured, and showed a distinct inhibition of transport in the case of the 54 kD protein.