Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.).

Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of <5, compared to the sweet cultivars that are generally characterized by mature fruit pH values of >6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit acidity within the species.

Pollution-affected fish hepatic transcriptome and its expression patterns on exposure to cadmium.

Individuals of the fish Lithognathus mormyrus were exposed to a series of pollutants including: benzo[a]pyrene, pp-DDE, Aroclor 1254, perfluorooctanoic acid, tributyl-tin chloride, lindane, estradiol, 4-nonylphenol, methyl mercury chloride, and cadmium chloride. Five mixtures of the pollutants were injected. Each mixture included one to three compounds. A microarray was constructed using 4608 L. mormyrus hepatic cDNAs cloned from the pollutant-exposed fish. Most clones (4456) were sequenced and assembled into 1494 annotated unique clones. The constructed microarray was used to identify changes in hepatic gene expression profile on exposure to cadmium administered to the fish by feeding or injections. Thirty-one unique clones showed altered expression levels on exposure to cadmium. Prominently differentially expressed genes included elastase 4, carboxypeptidase B, trypsinogen, perforin, complement C31, cytochrome P450 2K5, ceruloplasmin, carboxyl ester lipase, and metallothionein. Twelve sequences have no available annotation. Most genes (23) were downregulated and hypothesized to be affected by general toxicity due to the intensive cadmium exposure regime. The concept of an operational multigene cDNA microarray, aimed at routine and fast biomonitoring of multiple environmental threats, is outlined and the cadmium exposure experiment has been used to demonstrate functional and methodological aspects of the biomonitoring tool. The components of the outlined system include: (1) spotted array, composed of both pollution-affected and constitutively expressed genes, the latter are used for normalization; (2) standard, repeatable labeling procedure of a reference transcript population; and (3) biomarker indices derived from the profile of expression ratio across the pollution-affected genes, between the field-sampled transcript populations and the reference.

Hepatopancreatic multi-transcript expression patterns in the crayfish Cherax quadricarinatus during the moult cycle.

Alterations of hepatopancreatic multi-transcript expression patterns, related to induced moult cycle, were identified in male Cherax quadricarinatus through cDNA microarray hybridizations of hepatopancreatic transcript populations. Moult was induced by X-organ sinus gland extirpation or by repeated injections of 20-hydroxyecdysone. Manipulated males were sacrificed at premoult or early postmoult, and a reference population was sacrificed at intermoult. Differentially expressed genes among the four combinations of two induction methods and two moult stages were identified. Biologically interesting clusters revealing concurrently changing transcript expressions across treatments were selected, characterized by a general shift of expression throughout premoult and early postmoult vs. intermoult, or by different premoult vs. postmoult expressions. A number of genes were differentially expressed in 20-hydroxyecdysone-injected crayfish vs. X-organ sinus gland extirpated males.

Immunopotentiating and immunotherapeutic effects of thymic hormones and factors with special emphasis on thymic humoral factor THF-gamma2.

The essential role played by the thymus in the development of the immune response was well documented in many publications. These findings prompted a long series of studies devised to define the factors produced and secreted by thymus cells, which are involved in the development and nature of immunological responsiveness. First experiments done with crude thymus extracts were followed by isolation of purified products and finally by chemical characterization and synthesis of immunologically active thymus-derived peptides. In this article we review the various thymic hormones and factors described, that is, thymosin fractions 5, the thymosins, prothymosin alpha, thymulin (FTS-Zn), thymopoietin, thymostimulin (TP-1), Thymic humoral factor (THF), and THF-gamma2. Studies demonstrating the activity of the various thymic factors in increasing the immunocompetence potential in both in vitro and in vivo conditions are discussed. The immunostimulatory potential of thymic factors was also investigated in experimental models where beneficial therapeutic effects were sought in a situation of immunological malfunction. The last part of the review is dedicated to clinical trials with thymic factors that revealed improvement in the immunocompetence potential in cases of immunodeficiencies, viral infections, and cancer and its correlation with therapeutic effectiveness. It seems that more research is required in order to better define conditions for the use of thymic factors in immunotherapy.

A simple method for estimating the intensity of purifying selection in protein-coding genes.

We propose a method by which the intensity of purifying selection on a functional protein-coding gene is estimated by using three aligned homologous sequences: a processed pseudogene (psi), a functional paralog from the same species (g), and a functional ortholog from a different species (o). For each such trio, we calculate the numbers of nucleotide substitutions along the branches leading to psi and g, i.e., K psi and K(g). If we assume that the mutation rates are the same in the genes and the pseudogenes and that mutations occurring in a pseudogene do not affect the fitness of the organism, we can show that the fraction of mutations that are selectively neutral, fg, is equal to the ratio K(g)/K psi. Since advantageous mutations occur only very rarely, such that they do not contribute significantly to the rate of molecular evolution, the fraction of deleterious mutations that are subject to purifying selection is 1-fg. Therefore, the K(g)/K psi ratio can be used directly to estimate the intensity of purifying selection, thereby isolating its effects on the rate of evolution from those of mutation. We compared the selection intensities of 12 orthologous protein-coding pairs from humans and murids. As expected, the fraction of mutations that are subject to purifying selection is strongest in the second codon position and weakest in the third. Interestingly, the mean fractions of effectively neutral mutations in the third codon position were only 41% and 42% for murids and humans, respectively, indicating that many synonymous mutations are subject to selective constraint. In several orthologous genes, we found that the intensity of purifying selection is very different between murid and human orthologous genes. There was no statistically significant difference in overall intensity of purifying selection between humans and murids. Thus, purifying selection does not seem to be an important factor contributing to the observed differences in the rates of evolution between these two taxa.

THF-gamma 2-mediated reduction of pulmonary metastases and augmentation of immunocompetence in C57BL/6 mice bearing B16-melanoma.

Immunotherapy with the immunomodulating thymic humoral factor-gamma 2 (THF-gamma 2) octapeptide, combined with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy, will be used for enhancing host immune response to arrest pulmonary metastases of a B16-F10.9 melanoma tumor. In this experimental model of pulmonary metastasis, the highly metastatic B16-F10.9 melanoma tumor cells (2 x 10(5)) were inoculated into the footpad of mice to form a primary tumor. The tumor-bearing leg was surgically removed on reaching the size of 5.5 mm, which resulted in the appearance of metastases in the lungs of the animals. After tumor excision, mice were treated intraperitoneally with a single dose of BCNU (20 or 35 mg/kg) followed by a series of intraperitoneal THF-gamma 2 injections (1 microgram/0.5 ml/injection). Relative to untreated mice and those receiving chemotherapy alone, the antitumor action of the combined THF-gamma 2 chemoimmunotherapy protocol was significantly augmented according to the following in vivo parameters: (a) decreased postsurgical spontaneous metastatic burden; (b) prolonged survival time; (c) increased resistance to tumor cell challenge; and (d) massive infiltration of lymphocytes, polymorphonuclear cells, and macrophages in the lung tissue. The THF-gamma 2 immunotherapy also prevented a decrease in lymphocyte reactivity, otherwise induced by the tumor/BCNU chemotherapy. THF-gamma 2 immunotherapy resulted in restoration of the response to Lipopolysaccharide mitogenic stimulation and the allogeneic response. Our data suggest that postoperative THF-gamma 2 immunotherapy could be a valuable adjunct to anticancer chemotherapy as a treatment for metastatic arrest of melanoma tumor.

Constructing phylogenies from quartets: elucidation of eutherian superordinal relationships.

In this work we present two new approaches for constructing phylogenetic trees. The input is a list of weighted quartets over n taxa. Each quartet is a subtree on four taxa, and its weight represents a confidence level for the specific topology. The goal is to construct a binary tree with n leaves such that the total weight of the satisfied quartets is maximized (an NP hard problem). The first approach we present is based on geometric ideas. Using semidefinite programming, we embed the n points on the n-dimensional unit sphere, while maximizing an objective function. This function depends on Euclidean distances between the four points and reflects the quartet topology. Given the embedding, we construct a binary tree by performing geometric clustering. This process is similar to the traditional neighbor joining, with the difference that the update phase retains geometric meaning: When two neighbors are joined together, their common ancestor is taken to be the center of mass of the original points. The geometric algorithm runs in poly(n) time, but there are no guarantees on the quality of its output. In contrast, our second algorithm is based on dynamic programming, and it is guaranteed to find the optimal tree (with respect to the given quartets). Its running time is a modest exponential, so it can be implemented for modest values of n. We have implemented both algorithms and ran them on real data for n = 15 taxa (14 mammalian orders and an outgroup taxon). The two resulting trees improve previously published trees and seem to be of biological relevance. On this dataset, the geometric algorithm produced a tree whose score is 98.2% of the optimal value on this input set (72.1% vs. 73.4%). This gives rise to the hope that the geometric approach will prove viable even for larger cases where the exponential, dynamic programming approach is no longer feasible.

Experiential learning of clinical skills by beginning nursing students: "coaching" project by fourth-year student interns.

To fulfill the requirements for the internship program, fourth-year academic nursing students participated in a leadership program and became coaches for novice students who were beginning their first clinical rotations in the hospital. The concept of coaching is recognized in theory, research, and clinical education as an educational tool, which provides mutual benefits for the coachee and the coach. The project lasted 12 weeks, 2 clinical days per week. The coaches served as a source of support and knowledge and assisted in problem solving for the beginning students. As nurse educators who oversaw the project, the authors summarized the program as it was developed and implemented at the Assaf HaRofeh School of Nursing including problems, revisions, and final conclusions and discussion.

Patterns and rates of indel evolution in processed pseudogenes from humans and murids.

Patterns and rates of indel (deletions and insertions) evolution were characterized in 156 independently derived processed pseudogenes from humans and murids (mice and rats). A total of 441 deletions and 161 insertions were unambiguously identified. On a subset of 109 pseudogenes, we verified and confirmed the assumption that indels occur almost exclusively in the pseudogene and, therefore, in comparisons between pseudogenes and their functional paralogs, it is possible to assign polarity to the indel event. By comparing the characteristics of terminal truncations with those of internal deletions, we find support for the hypothesis that truncations are generated through a different pathway than internal deletions. The number of deletions and insertions per pseudogene was found to increase monotonically with time. Deletions occur on average once every 40 nucleotide substitutions, whereas insertions are much rarer, occurring once every 100 substitutions, indicating that the mechanisms involved in deletion formation are most probably different from those responsible for the formation of insertions. The age of the pseudogene, however, explained only 20 and 13%, respectively, of the variation in the number of deletions and insertions per site, indicating that factors other than evolutionary time may play a significant role in the evolutionary dynamics of indel accumulation. Since the rate of substitution has been previously shown to be higher in murids than in humans, we deduce that deletions and insertions accumulate proportionally faster in murids than in humans. Deletions and insertions in murid and human genomes do not contribute significantly to genome size.

Inhibition of murine Lewis lung carcinoma metastases by combined chemotherapy and intranasal THF-gamma 2 immunotherapy.

Previous research in our laboratories has shown that the immunoregulatory octapeptide, THF-gamma 2, potentiates the efficacy of anticancer chemotherapy in experimental animal models of local plasmacytoma and repairs drug-induced defects in immunocompetence. The highly metastatic, murine D122 lung carcinoma model has been shown to be useful for evaluating the efficacy of experimental antimetastatic therapeutic modalities. The goal of the present study was to determine whether intranasal thymic humoral factor-gamma 2 (THF-gamma 2) immunotherapy, after a single dose of chemotherapy, could inhibit the development of lung metastases, restore immunocompetence, and increase survival in syngeneic C57BL/6 mice bearing highly metastatic Lewis lung carcinoma (D122) solid footpad tumors. Relative to untreated mice and those receiving chemotherapy alone, mice receiving combined chemoimmunotherapy showed the following significant differences: (a) decreased lung metastatic load as assessed by lung weight, (b) prolonged survival time, (c) massive infiltration of lymphoid cells in the lungs, and (d) restoration of impaired immune parameters to normal values in melphalan-treated mice. THF-gamma 2 prevented tumor emboli from colonizing the target tissue, probably by inducing expansion of the lymphoid cell compartment. When used as an adjunct to anticancer chemotherapy, intranasal THF-gamma 2 immunotherapy is a simple and safe treatment modality that seems to be promising for inhibiting lung metastases.

Thymic humoral factor-gamma 2 (THF-gamma 2) immunotherapy reduces the metastatic load and restores immunocompetence in 3LL tumor-bearing mice receiving anticancer chemotherapy.

In mice bearing immunogenic tumors, adding thymic humoral factor-gamma 2 (THF-gamma 2)1 immunotherapy as an adjunct to anticancer chemotherapeutic regimens not only potentiates the antitumor activity of each drug but also repairs tumor/chemotherapy-induced damage to T-cell populations and functions. The Lewis lung carcinoma (3LL) is a weakly immunogenic, highly metastatic tumor in C57BL/6 mice. To investigate whether the immunoregulatory octapeptide is also effective against a tumor that does not elicit an antitumor immune response, we assessed the effect of combination THF-gamma 2 immunotherapy and chemotherapy in 3LL-bearing mice. The results indicate that THF-gamma 2 combined with either Melphalan or 5-Fluorouracil was more effective in reducing metastatic load than either chemotherapeutic drug alone and was characterized by massive infiltration of lymphatic cells. The combined chemoimmunotherapy treatment also prolonged the survival time in all treated animals and repaired T-cell defects and impaired in vitro cellular immune response parameters, induced either by the tumor or by chemotherapy. THF-gamma 2 immunotherapy reversed the decrease in the number of bone-marrow myeloid colonies (GM-CFU) induced by chemotherapy treatment of tumor-bearing mice, supporting the hypothesis that THF-gamma 2 directly stimulates the proliferation of myeloid stem cells. The overall results imply, that when administered as an adjunct to chemotherapy, THF-gamma 2 immunotherapy is equally effective against immunogenic and nonimmunogenic tumors.

Biased random mutagenesis of peptides: determination of mutation frequency by computer simulation.

Cassette mutagenesis is a method of protein engineering which generates a wide diversity of genetic variants that can be subjected to either selection or screening. As long as the target sequence to be modified is kept short (corresponding to four to six amino acids), complete combinatorial libraries can be produced. A major problem arises when longer peptides are to be engineered for desired functions. In such situations the production of a limited collection of variants can be helpful; thus, biased random mutagenesis and 'doping schemes' have been reported previously. Here we describe a computer algorithm that enables the determination of the degree of phosphoramidite contamination of nucleotide precursor reservoirs. Through simulation of biological translation, the algorithm allows the prediction of the effect of contamination levels on the number of mutations to occur for any given peptide sequence. In this study the cholinergic binding site was used as a model sequence (22 amino acids). Considerations, based on the computer program, are discussed regarding the efficient design of phage-display combinatorial libraries.

Release of immunosuppressive factor(s) by MOPC-315 murine plasmacytoma cells: a possible mechanism of defence.

Supernatants were collected from suspensions of MOPC-315 tumor cells harvested from ascitic tumors and kept for 24 hours in culture medium and from cultures of an MOPC-315 tumor-cell kept for a long period of time in vitro. The MOPC-315 supernatants were tested for immunosuppression of mitogenic stimulation of BALB/c spleen cells by ConA or LPS, of allogeneic response of effector BALB/c spleen cells against target C57BL spleen cells, of generation of antibody response against SRBC and of induction of LAK activity. The immunosuppression was marked in all the test systems, was not related to secretion of either C-type particles or of anti-TNP antibodies and was also induced by MOPC-315 tumor cells kept in serum-free medium. It is suggested that release of immunosuppressive factor(s) by MOPC-315 tumor cells might play a role in the mechanism(s) of defence of the tumor against the host.

Inhibition of chloroplast CF1-ATPase by vanadate.

Inhibition of ATPase activity by vanadate, having K1/2 of 0.5 mM, was demonstrated in the CF1-ATPase. The Ca(2+)-dependent ATPase activity of the isolated enzyme was inhibited in an allosteric manner by vanadate with a Hill coefficient of 3.19 +/- 0.6. Vanadate also inhibited ATPase and Pi-ATP exchange activities of the chloroplast membrane-bound enzyme. Using 51V NMR it was demonstrated that ATP caused partial release of about 1.87 equivalents while ADP caused additional binding of approximately 1.46 equivalents of vanadate, when added to a solution containing CF1 equilibrated with vanadate. The relevance of these results to a possible involvement of a pentacovalent phosphate as transition state intermediate in the hydrolysis of ATP by CF1-ATPase is discussed.

Therapeutic effectiveness against MOPC-315 plasmacytoma of low or high doses of the synthetic thymic hormone THF-gamma 2 in combination with an "immunomodulating" or a "non-immunomodulating" drug.

We reported previously that treatment of mice bearing MOPC-315 plasmacytoma with the drugs L-PAM (phenylalanine mustard) or 5-FU (5-fluorouracil), in combination with low doses of THF-gamma 2, was more effective in increasing their survival time than treatment with the drug alone. We show here that in the combined treatment using a single injection of 5-FU followed by multiple (8-15) injections of THF-gamma 2, the megadoses were more effective than the low doses in increasing the survival time of MOPC-315 tumor-bearing mice. On the other hand, in combination with L-PAM, both low and high doses of THF-gamma 2 were equally effective. The need for high doses of THF-gamma 2, when used in combination with 5-FU, could be due to the fact that 5-FU acts as a "non-immunomodulating" drug and has to be used at a high, immunosuppressive dose.

A synthetic thymic hormone, THF-gamma 2, repairs immunodeficiency of mice cured of plasmacytoma by melphalan.

BALB/c mice cured of large MOPC-315 plasmacytomas by melphalan remain deficient in their spleen T-cell functions. This was manifested by impairment of the allogeneic and the antibody responses in vitro to SRBC and in decreased numbers of T-cells including their subsets CD4 and CD8. IL-2 production and specific cytotoxicity against MOPC-315 tumor cells were, on the other hand, maintained. Treatment of these cured mice by in-vivo administration of THF-gamma 2, an octapeptide from calf thymus, repaired these deficits. This was evidenced by in vitro tests with spleen cells which manifested an increased allogeneic response and elevated generation of primary antibody response, restoration of T-cell subpopulations to normal and an enhanced IL-2 production above normal levels. The potential use of THF-gamma 2 as supportive therapy in cancer treatment is suggested.

Induction of protective antitumor immune response against a murine plasmacytoma not curable by alkylating-drug treatment.

Immunization with viable tumor cells followed by subsequent administration of glutaraldehyde-treated tumor cells induced a protective antitumor immune response in the host toward the alkylating-drug resistant RPC-5 plasmacytoma. This was proven by resistance to challenge with RPC-5 tumor cells, neutralization in Winn tests, by effectiveness of combined chemotherapy with melphalan plus immunotherapy with spleen cells from RPC-5 immunized mice and in vitro by cytotoxicity tests. The specificity of the immune response was ascertained in vivo by comparison with the response toward MOPC-315 plasmacytoma. However, in vitro cytotoxicity tests revealed the occurrence of shared antigens between the RPC-5 and MOPC-315 tumor cells. It is concluded that the ineffectiveness of alkylating-drug treatment toward the RPC-5 tumor is not due to the inability of this tumor to induce a specific antitumor immune response, and that cross-antigenic relationship as revealed by in vitro cytotoxicity tests does not necessarily reflect cross-protection between various plasmacytomas.

THF-gamma 2, a synthetic thymic hormone, increases effectiveness of combined chemotherapy and immunotherapy against RPC-5 murine plasmacytoma.

The effect of a synthetic thymic hormone, THF-gamma 2, on the anti-tumor activity of spleen cells was studied in mice immunized against the RPC-5 tumor. Following two courses of the THF-gamma 2 treatment, the mean RPC-5 specific cytotoxic response of immune spleen cells was significantly increased when compared to normal cells (P less than 0.001) and to untreated immune spleen cells (P less than 0.04). In addition, THF-gamma 2 treatment improved the competence of immune spleen cells in adoptive immunotherapy (AIT) when performed in combination with chemotherapy by melphalan. Recipients of spleen cells from THF-gamma 2 treated mice showed a 35% increase in survival when compared to AIT with immune cells alone. The results suggest that THF-gamma 2 treatment of donors for AIT might be applicable to cancer therapy in humans.

Immunocompetence status as related to growth progression of mouse MOPC-315 plasmacytoma.

The immunocompetence status of mice bearing MOPC-315 plasmacytoma was determined at various days after tumor inoculation. Changes in T and B-cell functions appeared gradually. The allogeneic response of spleen cells from BALB/c tumor-bearing mice against C57BL spleen cells was impaired from the 4th day after the tumor inoculation (nonpalpable tumor stage). The primary antibody response in vitro against SRBC was depressed at 18 days, and the mitogenic response of splenic cells to PHA and to LPS was depressed at 25 days after the tumor inoculation. T cells taken from day 18 tumor-bearing mice partially suppressed the MLR response of normal splenocytes. Mice bearing large MOPC-315 tumors responded less to SRBC immunization than normal, noninoculated mice. The relative percentage of Lyt 1, Lyt 2 and L3T4 T-cell subsets decreased starting from the 11th day after tumor inoculation.

THF-gamma 2, a thymic hormone, increases immunocompetence and survival in 5-fluorouracil-treated mice bearing MOPC-315 plasmacytoma.

The effect of the thymic hormone, THF-gamma 2, on the immunocompetence of 5-fluorouracil (5-FU)-treated BALB/c mice, bearing MOPC-315 tumor, was examined. Treatment of noninoculated or tumor-bearing mice with THF-gamma 2 after 5-FU injection, resulted in an increase in the antibody response to sheep red blood cells and in the allogeneic response in spleen cell cultures and had no effect on the concanavalin-A-induced interleukin-2 secretion beyond that caused by 5-FU alone. Treatment with either 5-FU alone or 5-FU and THF-gamma 2 resulted in restoration to normal values of Lyt1- and L3T4-positive populations in tumor-bearing mice. THF-gamma 2 prolonged the survival time of mice bearing MOPC-315 tumor beyond that observed in mice treated with 5-FU alone.