Grafting Tomato to Manage Bacterial Wilt Caused by Ralstonia solanacearum in the Southeastern United States.

Bacterial wilt, caused by Ralstonia solanacearum, can result in severe losses to tomato (Solanum lycopersicum) growers in the southeastern United States, and grafting with resistant rootstocks may be an effective strategy for managing this disease. However, R. solanacearum populations maintain considerable diversity, and little information is known regarding the efficacy of commercially available rootstocks to reduce bacterial wilt incidence and subsequent crop loss in the United States. In this study, tomato plants grafted onto 'Dai Honmei' and 'RST-04-105-T' rootstocks had significantly lower area under the disease progress curve (AUDPC) values compared with nongrafted plants (P < 0.05). Across three locations in North Carolina, final bacterial wilt incidence for non- and self-grafted plants was 82 ± 14 to 100%. In contrast, bacterial wilt incidence for the grafted plants was 0 to 65 ± 21%. Final bacterial wilt incidence of plants grafted with Dai Honmei rootstock was 0 and 13 ± 3% at two locations in western North Carolina but 50 ± 3% at a third site in eastern North Carolina. Similarly, grafting onto RST-04-105-T rootstock significantly reduced AUDPC values at two of the three locations (P < 0.05) compared with that of the nongrafted plants, but performed poorly at the third site. Total fruit yields were significantly increased by grafting onto resistant rootstocks at all three sites (P < 0.05). Regression analyses indicated that yield was significantly negatively correlated with bacterial wilt AUDPC values (R2 was 0.4048 to 0.8034), and the use of resistant rootstocks enabled economically viable tomato production in soils naturally infested with R. solanacearum.

Acquisition of Key-Pecking via Autoshaping as a Function of Prior Experience: "Learned Laziness"?

A group of pigeons that had previously received noncontingent food delivery acquired the key-peck response (in autoshape training) more slowly than did a naive control group; key-peck acquisition was most rapid for a group given operant treadle-press training in the initial phase.

Enhancing activity of various immunoaugmenting agents on the delayed-type hypersensitivity response in mice.

Six immunoaugmenting agents were tested in the delayed-type hypersensitivity reaction (DTH) in normal BALB/c X DBA/2 mice. The agents tested, levan, lentinan, mannozym, maleic anhydride divinyl ether, polyriboinosinic-polycytidylic acid-poly-L-lysine, and highly purified L-cell interferon, gave significant increases in the DTH response above the sheep red blood cell control. The schedule of doses for each agent corresponded with previous experiments from this laboratory of the maximum natural killer cell activity, macrophage activation, and interferon induction. Highly purified L-cell interferon was capable of eliciting a significant DTH response when given 4 hr after the initial challenge with sheep erythrocytes. In addition, lambda-carrageenan, a macrophage-cytotoxic agent which can render the macrophage inactive, was found to suppress the DTH response to levels slightly above phosphate-buffered saline controls. The carrageenan-induced suppression of the DTH response could be abrogated by coadministration with immunoaugmenting agents to levels attained with the immunoaugmenting agents alone.

In vivo modulation of myelopoiesis and immune functions by maleic anhydride divinyl ether copolymer (MVE-2) in tumor-free and MBL-2 tumor-bearing mice treated with cyclophosphamide.

Treatment of normal or MBL-2 tumor-bearing mice with cyclophosphamide (CY) caused severe suppression of myelopoiesis and macrophage (M phi) functions, both of which may limit further use of chemotherapy. Additional treatment with the chemically defined biological response modifier maleic anhydride divinyl ether copolymer (MVE-2) was able to ameliorate the myelosuppressive effects of CY and to restore normal bone marrow cellularity. The stimulatory effects on myelopoiesis, however, could only be obtained by administering MVE-2 at greater than or equal to 3 days after CY, which correlated with an MVE-2-induced simultaneous increase in granulocyte and/or macrophage colony-stimulating factor secretion by bone marrow cells or M phi. Injection of MBL-2 tumor-bearing mice with MVE-2, at 3 days after Cy treatment, caused a decrease in tumor burden and a significant increase in median survival time as compared to treatment with CY alone. At the same time, MVE-2 induced an increase in the number of cytotoxic M phi and a complete restoration within the myelopoietic lineage, which might prevent delayed side effects of CY, such as secondary infections, and might permit more intensive chemotherapeutic treatment. Treatment of MBL-2 tumor-bearing mice with MVE-2, at 6 days after CY, induced a significant increase in M phi cytotoxicity but did not prolong median survival time, probably due to a rapid regrowth of tumor after treatment with CY. Our studies thus show that successful combined therapy with the primary cytotoxic agent CY and the biological response modifier MVE-2 depends on precise timing of the drug regimen and is influenced by the extent and reversibility of CY-induced immunosuppression, as well as by the kinetics of recruitment of new effector cells from bone marrow and by the tumor burden present at the time of treatment.

Interpersonal concomitants and antecedents of depression among college students.

Depressed college students were compared with other-psychopathology and normal controls regarding the relationship they developed with dormitory roommates during a 9-month period. Diagnostic status was periodically assessed via SADS interviews, thus also permitting identification of new cases of depression during the year. Psychosocial characteristics found to be uniquely associated with current depression were: (a) low social contact with roommates, (b) low enjoyability of these contacts, and (c) high life-event stress. Roommates of depressives reported low enjoyability of the relationship and high levels of aggressive behavior towards the depressive. No features were found to be uniquely associated with new cases before they became depressed; however, several antecedents of general psychopathology were identified.

Abstraction of themes from melodic variations.

A schema formation analysis was applied to listeners' abstraction of themes from melodic variations. A set of transformations of a prototypical melody was generated by systematic application of five transformational rules. The prototype represented the structural central tendency of the set. In Experiment 1 participants listened to the transformations and were tested for abstraction of the prototype in two ways; (a) they were asked to draw the melodic contour of 10 notes best describing the central tendency of the set of transformations, and (b) they were given a false recognition task in which they were asked to recognize the prototype and other transformations, none of which were in the original set, in a procedure analogous to that used by Franks and Bransford (1971). Both measures indicated that participants abstracted the prototype while listening to the original set of transformations. Experiment 2 replicated these findings with participants fully informed about the purpose and design of the experiment, a different task for recognizing the prototype (judging the similarity of transformations to the abstracted prototype), and a different prototype. In Experiment 3 participants who listened to the prototype produced data on the contour drawing and recognition tasks that were indistinguishable from those produced by participants who abstracted the theme from its variations.

Responses to depressed interpersonal behavior: mixed reactions in a helping role.

We placed 144 female subjects in a helping role and randomly assigned them to interact with a confederate in a 3 X 3 X 2 X 2 (Psychopathology X Blaming X Advice Seeking X Sex of Confederate) factorial design. In order to study behaviors that mediate interpersonal responses to depression, male and female confederates enacted depressed, anxious, or normal roles and blamed themselves, others, or no one for their problems. The confederates requested advice in half of the conditions. Results indicated that depressed confederates were rejected more on questionnaire measures; however, depressed confederates received more conversational advice and support from subjects than did the equally disturbed anxious confederates. The self-blaming and advice-seeking manipulations did not interact with depression to produce more negative reactions in subjects. There was no evidence of a negative mood induction in subjects, nor did the sex of the confederate have important interpersonal consequences. Results are discussed in terms of theoretical and methodological issues in studies of interpersonal factors in depression.

Update on myocardial contrast echocardiography: a surgeon's perspective.

The ability to evaluate myocardial perfusion and microvascular structural integrity can help surgeons predict the necessity for surgical intervention, the sequence of intraoperative interventions, the risk of perioperative infarction, the likelihood of successful surgical recovery, and the degree of long-term clinical benefit. The ability to directly assess perfusion intraoperatively may allow surgeons to reliably evaluate a patient's myocardial perfusion at any time during the operative procedure. As this article will discuss, surgeons may use myocardial contrast echocardiography intraoperatively to evaluate myocardial function and integrity, to determine the order of graft placement, to determine the success of bypass graft patency, and to help predict those patients who will experience successful cardiac function after recovering from surgery.

Lyme disease acquired in Europe and presenting in CONUS.

Lyme disease is recognized in many parts of the world, including large areas of North America, Europe, Asia and Australia. Diagnosis and treatment of the disease is essential to avoid the debilitating and potentially life-threatening long-term effects of the infection; however, many physicians may not be aware of the international scope of the disease. This is particularly important for military physicians whose patients may visit or live in endemic areas and whose activities may bring them in contact with the organism. We report here the case of a soldier with near-fatal Lyme carditis acquired in Europe and presenting in Massachusetts.

Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore.

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.

Plasma membrane targeting of chimeric intracisternal A-type particle polyproteins leads to particle release and specific activation of the viral proteinase.

Retrovirus morphogenesis involves assembly of structural Gag polyproteins with subsequent budding from the plasma membrane, followed by proteolytic cleavage by the viral proteinase (PR) and extracellular maturation to the infectious virion. Intracisternal A-type particles (IAPs) are defective retroviruses that assemble and bud at the membranes of the endoplasmic reticulum (ER), where they remain as immature particles consisting exclusively of uncleaved polyproteins. To analyze requirements for intracellular polyprotein transport and PR activation, we constructed deletion and substitution mutations in the IAP gag gene, including the putative ER-targeting signal. Mutant polyproteins were transported to various intracellular locations, including the nucleus, the cytoplasm, the ER, and the plasma membrane. Interestingly, assembly of capsid-like particle structures occurred at almost all sites. However, only those polyproteins transported to the plasma membrane were efficiently and specifically cleaved by viral PR, with cleavage occurring predominantly within the virus particle. Thus, at least in the experimental system presented here, retroviral particle assembly can occur at almost any location within the cell, while polyprotein processing and, consequently, virion maturation are confined to a specific cellular site. These results suggest that a factor restricted to the plasma membrane is required to trigger PR activation and maturation of infectious retroviruses.

Intracisternal A-type particles express their proteinase in a separate reading frame by translational frameshifting, similar to D-type retroviruses.

Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum of rodent cells. IAP genomes share extensive sequence homologies with D-type retroviruses, but were presumed to express the viral proteinase (PR) as part of the gag open reading frame (ORF) while D-type retroviruses express PR in a separate ORF. Here we show that expression of the murine IAP element MIA14 yields three major translation products, corresponding to the Gag, Gag-PR, and Gag-PR-Pol polyproteins. Sequence analysis revealed that MIA14 PR is encoded in its own reading frame, separate from gag and pol. Frameshifting occurred with an efficiency of approximately 25% between the gag and pro ORFs and 35% between pro and pol. The region containing the putative gag-pro frameshift signal consists of a heptanucleotide slippery sequence (A6C) and a stem-loop structure probably forming a pseudoknot. Deletion of this structure element almost completely abolished frameshifting. Insertion of an additional base next to the frameshift signal placed gag and pro in the same ORF and resulted in predominant formation of Gag-PR and Gag-PR-Pol polyproteins which were not processed following in vitro translation. Expression of a similar construct in tissue culture cells, on the other hand, led to efficient intracellular processing of the mutant polyproteins.

Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity.

The nef gene of primate immunodeficiency viruses is essential for high-titer virus replication and AIDS pathogenesis in vivo. In tissue culture, Nef is not required for human immunodeficiency virus (HIV) infection but enhances viral infectivity. We and others have shown that Nef is incorporated into HIV-1 particles and cleaved by the viral proteinase. To determine the signal for Nef incorporation and to analyze whether virion-associated Nef is responsible for enhancement of infectivity, we generated a panel of nef mutants and analyzed them for virion incorporation of Nef and for their relative infectivities. We report that N-terminal truncations of Nef abolished its incorporation into HIV particles. Incorporation was reconstituted by targeting the respective proteins to the plasma membrane by using a heterologous signal. Mutational analysis revealed that both myristoylation and an N-terminal cluster of basic amino acids were required for virion incorporation and for plasma membrane targeting of Nef. Grafting the N-terminal anchor domain of Nef onto the green fluorescent protein led to membrane targeting and virion incorporation of the resulting fusion protein. These results indicate that Nef incorporation into HIV-1 particles is mediated by plasma membrane targeting via an N-terminal bipartite signal which is reminiscent of a Src homology region 4. Virion incorporation of Nef correlated with enhanced infectivity of the respective viruses in a single-round replication assay. However, the phenotypes of HIV mutants with reduced Nef incorporation only partly correlated with their ability to replicate in primary lymphocytes, indicating that additional or different mechanisms may be involved in this system.

Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells.

Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pol open reading frame did not yield particles.

Immunosuppressive activity of human chorionic gonadotrophin preparations in vivo: evidence for gonadal dependence.

Human chorionic gonadotrophin preparations (hCG), when injected ip daily for 4 days, suppress the delayed-type hypersensitivity (DTH) response of mice to sheep red blood cells. Preparations of crude hCG, purified hCG subunits, and hCG that was formed by recombining the purified subunits showed immunosuppressive activity in accord with their gonadotrophic activity. The immunosuppressive effects in male and female mice were comparable. However, removal of the gonads completely abrogated the immunosuppressive activity of hCG in both males and females, suggesting that the effect of hCG is mediated by a factor released from the gonads. We conclude that the hCG molecule itself exhibits immunosuppressive activity in vivo in both male and female mice and that the gonads are required for the expression of this activity.

Biochemical and structural analysis of isolated mature cores of human immunodeficiency virus type 1.

Mature human immunodeficiency virus type 1 (HIV-1) particles contain a cone-shaped core structure consisting of the internal ribonucleoprotein complex encased in a proteinaceous shell derived from the viral capsid protein. Because of their very low stability after membrane removal, HIV-1 cores have not been purified in quantities sufficient for structural and biochemical analysis. Based on our in vitro assembly experiments, we have developed a novel method for isolation of intact mature HIV-1 cores. Concentrated virus suspensions were briefly treated with nonionic detergent and immediately centrifuged in a microcentrifuge for short periods of time. The resuspended pellet was subsequently analyzed by negative-stain and thin-section electron microscopy and by immunoelectron microscopy. Abundant cone-shaped cores as well as tubular and aberrant structures were observed. Stereo images showed that core structures preserved their three-dimensional architecture and exhibited a regular substructure. Detailed analysis of 155 cores revealed an average length of ca. 103 nm, an average diameter at the base of ca. 52 nm, and an average angle of 21.3 degrees. There was significant variability in all parameters, indicating that HIV cores are not homogeneous. Immunoblot analysis of core preparations allowed semiquantitative estimation of the relative amounts of viral and cellular proteins inside the HIV-1 core, yielding a model for the topology of various proteins inside the virion.