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Tomato ( Solanum lycopersicum L.) is an important crop that possesses about 35,000 genes. The treatment of plants with elicitors or pathogen attacks causes a cascade of defense reactions. We investigated tomato responses to the BamFX TM solution containing Zn and Cu elicitors and report the results of comparative transcriptome analysis of tomato seeds treated with Zn and Cu elicitors. The seeds were treated with optimum concentrations of Bam-FX solutions and subjected to cold methanolic extraction methods to obtain the secondary metabolites produced within them at different time intervals post-Bam-FX treatment. The metabolite mixture was analyzed using gas chromatography-mass spectrometry (GCMS). In transcriptome sequencing, GO and KEGG analyses revealed that the majority of the DEGs in BamFx-treated tomato was associated with primary and secondary metabolism, plant hormone signal transduction, TF regulation, transport, and responses to stimuli.The secondary metabolites found in the BamFX treated tomato seedlings - Esters of Fumaric acid, Succinic acid etc. The transcript levels of most auxin transporter-encoding genes changed significantly in the BamFX-treated seedlings (e.g., Solyc01g007010.3, a RING-type E3 ubiquitin transferase). The gene Solyc07g061720.3 for Gibberellin 2-oxidase and the Phorbol-ester/DAG-type domain-containing protein (Solyc02g068680.1) associated with the intracellular signaling genes were found upregulated in the BamFx-treated seeds. The time-dependent effect of the BamFX (1:500 for 60 min) was found to be regulating Abscisic acid signaling pathway genes (Solyc09g015380.1). This study identified many candidate genes for future functional analyses and laid a theoretical foundation for an improved understanding of the molecular mechanisms involved in the BamFx treatment of tomatoes to improve stress resistance.

Although highly active antiretroviral therapy (HAART) has resulted in remarkable decline in the morbidity and mortality in AIDS patients, controlling HIV infections still remain a global health priority. HIV access to the CNS serves as the natural viral preserve because most antiretroviral (ARV) drugs possess inadequate or zero delivery across the brain barriers. Thus, development of target-specific, effective, safe, and controllable drug-delivery approach is an important health priority for global elimination of AIDS progression. Emergence of nanotechnology in medicine has shown exciting prospect for development of novel drug delivery systems to administer the desired therapeutic levels of ARV drugs in the CNS. Neuron-resuscitating and/or antidependence agents may also be delivered in the brain through nanocarriers to countercheck the rate of neuronal degradation during HIV infection. Several nanovehicles such as liposomes, dendrimers, polymeric nanoparticles, micelles, and solid lipid nanoparticles have been intensively explored. Recently, magnetic nanoparticles and monocytes/macrophages have also been used as carrier to improve the delivery of nanoformulated ARV drugs across the blood-brain barrier. Nevertheless, more rigorous research homework has to be elucidated to sort out the shortcomings that affect the target specificity, delivery, release, and/or bioavailability of desired amount of drugs for treatment of neuroAIDS., (Copyright © 2014 John Wiley & Sons, Ltd.)

DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP(C)) 23-231 by systematic evolution of ligands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 10(24) distinct nucleic acid species. Sixty nanograms of rhuPrP(C)23-231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrP(C)23-231 at 10(-6) M to 10(-8) M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP(C)90-231. Theoretical thermodynamic modeling of selected aptamer sequences identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrP(C)23-231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrP(C) expressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrP(C) specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrP(C) and mammalian PrP(C) with varying affinities and can be applied to biological samples for PrP(C) enrichment and as diagnostic tools in double ligand assay systems.

This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5' untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634-639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.

In this study we examined the in vitro effects of alcohol on the proliferative responses of lymphocytes from healthy donors and AIDS patients to a recombinant fusion peptide, env-gag, corresponding to portions of the gp41 envelope (env) and internal core (gag) proteins of HIV. The effects of alcohol (ETOH) on the natural killer (NK) cell activities of lymphocytes from healthy donors and patients with AIDS were also investigated. Peripheral blood mononuclear cells from both normal donors and AIDS patients produced significant levels of lymphocyte proliferative responses to the HIV env-gag peptide; however, these responses were significantly higher in patients with AIDS, showing the specificity of the response. The env-gag-induced proliferative responses of lymphocytes from normal subjects were significantly suppressed when cultures contained only higher levels of ETOH (0.2% and 0.3%), whereas ETOH even at a lower level (0.1%) produced significant suppression of the env-gag-induced proliferation of lymphocytes only from AIDS patients. Direct addition of ETOH at concentrations of 0.1%, 0.2%, and 0.3% to cultures of lymphocytes from normal donors and NK target cells did not produce significant suppression of NK cell activities. However, ETOH at concentrations of 0.2% and 0.3% significantly suppressed the NK activities of lymphocytes from AIDS patients, and the suppressive effect was observed at all E:T cell ratios examined. Control peptide from the Escherichia coli expression vector did not produce any significant effect on lymphocyte proliferative responses or NK activity of both normal donors and AIDS patients.(ABSTRACT TRUNCATED AT 250 WORDS)

To simplify our procedure for blood sample processing for PCR, we introduced a simpler, shorter, and more cost-effective method for the separation of peripheral blood lymphocytes (PBL) and DNA extraction for amplification with Taq polymerase. By this method, blood samples are processed in two simple 15-min steps: (1) separation of PBLs from whole blood by red blood cell lysis with the Roche Specimen Washing Solution, and (2) DNA extraction by heat-detergent treatment of separated PBLs. This new method is simpler than the standard Ficoll-Hypaque method for PBLs separation and Proteinase K digestion for DNA extraction. It is not inhibitory to DNA amplification and it allows effective processing of blood samples even after prolonged storage (as long as 8 days) at room temperature.

Replication in vitro of murine cytomegalovirus was found to be enhanced in lymphoid cells from leukemic (AKR/J) mice as compared with similar cells from nonleukemic animals. Prolonged productive murine cytomegalovirus infection in lymphoid organs in vivo was demonstrable only in leukemic AKR/J mice. Latent nonproductive murine cytomegalovirus infection established in young nonleukemic AKR/J mice was invariably reactivated and expressed in salivary glands and lymphoid organs after these animals became leukemic.

Pregnant rabbits of two partially inbred strains, WH/J and IIIVO/J, were given ten consecutive daily ip injections of 10 mg N-ethyl-N-nitrosourea (ENU)/kg dissolved in trioctanoin starting on either day 10, 15, or 18 of gestation. Of 7 WH/J progeny weaned, 3 developed primary renal tumors at 6.8 +/- 0.6 months of age (mean +/- SE). Similarly, of 12 IIIVO/J progeny weaned, 10 developed primary renal tumors at 6.3 +/- 0.6 months of age (mean +/- SE); in addition, 1 fibroblastic osteosarcoma and 5 neurofibromas, some associated with neurilemma cysts, were observed. Renal tumors were nephroblastomas , which appeared to develop within small renal cortical cysts. The frequency of tumor induction for each strain was similar to that in our previous experiments with acute administration of ENU and ethyl-urea plus NaNO2, but the latency period (6.4 +/- 0.5 mo) was almost doubled. In addition, chronic treatment of ENU induced teratogenic effects. In a number of these progeny, a disproportionate stunting or miniaturization was evident, the frequency of which increased with the age of the treated fetuses. In contrast, holes were found in the parietal bones at birth, but with a frequency that decreased with increasing fetal age at time of treatment. No teratogenic effects were observed in the controls.

A clone of L1210 mouse leukemia cells selected for resistance to both the antiviral and anticellular properties of mouse interferon were essentially devoid of fatty acid cyclooxygenase activity. Experiments in which broken cell preparations were mixed or the two cell types were cultivated together failed to indicate the presence of a diffusible enzyme inhibitor. Fatty acid lipoxygenase activity of similar magnitude was detectable in both cell types. A selective impairment of fatty acid cyclooxygenase in interferon-resistant cells is consistent with recently described data suggesting that this enzyme may play a crucial role in mediating the antiviral and anticellular effects of interferon.

We describe the antitumor effects of a certain RNA(s) isolated from murine tumors and embryos. A single i.v. injected of 10 to 30 microgram of this RNA(s) induces necrosis, hemorrhages, and regression of solid tumors in the strain of origin; in pregnant mice it is embryotoxic, causing resorption of the embryos, but has no toxic effect upon the tumor-bearing or pregnant host. Its action is highly specific, and its presence in both tumors and embryos suggests that it may result from reexpression of embryonic genes in tumor cells.

Synchronized entry of Vesicular Stomatitis Virus (VSV) into spin labeled cultured human cells resulted in an increase in the rigidity of cell membranes as measured by Electron Spin Resonance Spectroscopy. Treatment of spin labeled cells with homologous interferon alpha did not influence the membrane fluidity, neither did it significantly prevent the VSV induced membrane changes despite its anti-viral protection.

Human immunodeficiency virus (HIV) is the etiological agent in acquired immunodeficiency syndrome (AIDS). The presence of antibodies against HIV is indicative of prior exposure to HIV. Currently available antibody screening tests use HIV-infected cells as a source of viral antigens. This approach has resulted in inherent flaws, including a high rate of false-positive results. In order to develop a highly specific and sensitive test, various proteins of HIV [envelope (env), gag, pol] were cloned and expressed. In addition, we synthesized peptides corresponding to various regions of envelope (gp120, gp41), tat, art, and the protease gene products were synthesized. Using these synthetic peptides and cloned proteins, several conserved and highly antigenic epitopes in viral envelope were identified. Fusion proteins were made from the highly conserved and antigenic epitopes of env and gag. One of these fusion proteins, env-gag, was used to develop an enzyme immunoassay that would detect the antibodies against both env and gag epitopes. A clinical study of 13,781 samples demonstrated the very high specificity (99.9%) and sensitivity (100%) of this assay.

Interferon exhibits pleotropic effects on homologous cells. Interferons may be used clinically for both antiviral and antitumor therapy. A better understanding of how interferon achieves its hormonal effects should be useful in developing more judicious and specific applications of these natural substances in therapy. Interferon induces increased activity of two enzymes, 2'5'-oligoadenylate synthetase and a protein kinase, that depend on double-stranded RNA for activation. 2'5' A polymerizes ATP into a novel 2'5'-linked oligonucleotide, which in turn can activate a latent cellular nuclease (RNase L) which degrades mRNA. The second dsRNA-dependent enzyme, a protein kinase, phosphorylates a protein of approximately 67,000 daltons as well as the small subunit of eukaryotic initiation factor (eIF-2). The phosphorylation of eIF-2 results in the inhibition of protein synthesis. The extent of sensitivity to exogenous interferon could be influenced by several factors, including the number of cell surface receptors for interferon and the rate and efficiency at which ligand binding and "processing" is achieved. Prostaglandins, cyclic nucleotides, and oxygen-free radicals could participate in modulation of interferon action at this level. Interferon induces specific changes in the composition of membrane lipids. These changes included loss of unsaturated fatty acids from phospholipids, significant increases in levels of unesterified fatty acids, and moderate increases in concentrations of triglycerides and cholesterol esters. The changes were absent in cells treated with interferon in the presence of inhibitors of fatty acid cyclooxygenase or superoxide dismutase. Whether or not interferon-associated lipid changes directly participate in interferon action is not yet clear. Apparent diversity in its mechanistic approach to virus inhibition may be the key to the success of interferon as a wide-spectrum antiviral agent. Heterogeneity in molecular species of interferon may signify its molecular evolutionary adaptations to specific needs and the eventual development of a "fool-proof" system that we now perceive as the "interferon-system." The observed pleotropic effects may be due to linkage to a broader cellular machinery that operates to identify and effectively handle "foreign" substances.

Sera obtained from normal subjects and juvenile-onset diabetes (JD) and systemic lupus erythromatus (SLE) patients were examined for free fatty acid composition and 6-keto-PGF1 alpha content. In addition, prostaglandins in urine samples from normal and diabetic individuals were separated by HPLC, and 6-keto-PGF1 alpha levels were monitored by RIA. Arachidonate (20:4) content in diabetic and SLE individuals were significantly lower than that of controls. Urine from diabetic individuals showed decreased levels of 6-keto-PG F1 alpha. The study also indicated that RIA measurements on crude biological samples may yield erroneous data due to immune cross reactivity with other compounds.