<i>Streptomyces</i> PRIO41 as plant growth promoter of jalapeño pepper plants and as biocontrol agent of <i>Fusarium</i>

Chili pepper is one of the main crops of economic importance in Mexico, and Fusarium wilting is a disease that limits its production. In addition, the inappropriate use of agrochemicals in farming activities generate environmental and health problems. Therefore, in this study the effectiveness of Streptomyces sp PRIO41 was evaluated as a (1) biocontrol agent of Fusarium spp and (2) plant growth promoter bacteria. Assays of pathogenicity and virulence of Fusarium spp. in jalapeño pepper seeds, and interactions of these pathogens with Streptomyces PRIO41 were evaluated under two nutritional conditions. In the greenhouse, the effectiveness of Streptomyces sp. PRIO41 was determined as a (1) biocontrol of Fusarium, and (2) plant growth promoter of wilt of pepper plants. The results showed that all fungal isolates caused symptoms in pepper seeds and seedlings with different degrees of virulence. Interactions in vitro showed that Streptomyces showed the most effective range of virulence against Fusarium isolates in the poor medium (37.6%-100%), with fungicidal effects in some cases. In the greenhouse, Streptomyces PRIO41 reduced Fusarium wilting up to a 40%, and positively affected all vegetative growth parameters, particularly plant height, leaf area, root length, and leaf and root dry biomasses. This study showed the potential of Streptomyces PRIO41 as a biocontrol agent of Fusarium spp., and as a biofertilizer of pepper plants.     

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.      

Proteolytic conversion of insulin-like growth factors to an acidic form(s).

The relative amounts of the various forms of bioassayable insulin-like growth factors (IGF) isolated from human serum or serum fraction Cohn IV-1 depend on the purification procedure. With acid gel filtration or acid/ethanol extraction as the initial step, IGF-II (pI approximately 6.5) was the most abundant (40-70%) followed by somatomedin A (pI approximately 7.4; 15-23%), an acidic form of insulin-like activity (ILA pI 4.8) (13-21%) and IGF-I (pI approximately 8.5; 5-27%). If, however, pH 5.5 ion-exchange chromatography on SP-Sephadex was used prior to acid gel filtration, the acidic pI 4.8 form was the major (greater than 90%) species recovered and was accompanied by a quantitative loss of the other IGF species. This suggested a possible conversion of IGF-I, somatomedin A and/or IGF-II to the acidic ILA pI 4.8 form(s) during the SP-Sephadex procedure. Further experiments indicated that differences in the yields of ILA pI 4.8 were not due simply to differences in the initial pH conditions of the various methods (i.e. acid versus neutral), although exposure to pH 9.7 (a pH experienced during elution of IGF activity from the SP-Sephadex) did appear to play a role. The involvement of the carrier protein in the conversion process was tested by subjecting carrier-free IGF-I and IGF-II to the SP-Sephadex procedure. No conversion of the free forms to ILA pI 4.8 occurred. To examine the possible role of proteinase in the conversion of IGFs to ILA pI 4.8, SP-Sephadex chromatography was performed in the presence of a broad spectrum proteinase inhibitor. The IGF distribution pattern obtained closely resembled the 'normal' pattern seen with acid gel filtration, indicating that proteinase inactivation had prevented conversion to ILA pI 4.8. These data suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to more acidic ILA pI 4.8 form(s) (i) occurs during SP-Sephadex chromatography, (ii) is not prevented simply by prior acid exposure, and (iii) takes place only when IGF-I and -II are in their high-Mr carrier-bound forms. Since IGF-I and IGF-II, although homologous, have unique amino acid sequences, the conversion of both IGFs implies that at least two acidic ILA forms exist. Nevertheless, because ILA pI 4.8 retains the full spectrum of IGF bioactivities in vitro, and significant quantities are present in normal human serum (21%), it would suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to ILA pI 4.8 in vivo may be a physiologically significant event.     

Insulin-like growth factor characteristics of an acidic non-suppressible insulin-like activity.

The biological activities of an acidic form of non-suppressible insulin-like activity (ILA pI 4.8) have been studied. ILA pI 4.8 was isolated from Cohn fraction IV-1 of human serum by pH 5.5 ion-exchange chromatography on SP-Sephadex. Carrier-bound ILA was eluted at pH 9.7 and then sequentially gel chromatographed in 1% formic acid on Sephadex G-75 and Bio-Gel P-30. The low-Mr (7000) active material was subjected to flat bed isoelectric focusing. Overall recovery was 87 munit of insulin equivalents/100 g of Cohn fraction IV-1, with a specific activity in the range 4-10 munit/mg of protein, representing a purity of 1-6%. This material has been tested in a variety of insulin-like growth factor (IGF)/somatomedin assay systems. It stimulated, in a dose-related manner, [14C]glucose conversion into lipid by isolated rat adipocytes, 35SO4(2-) incorporation into weanling rat costal cartilage and [3H]thymidine incorporation into DNA of cultured human fibroblasts. Like IGF-I and -II, ILA pI 4.8 was able to inhibit degradation of 125I-insulin by crude homogenates of rat liver. In addition, the biological activity of ILA pI 4.8 was completely suppressible by a recently described inhibitor of IGF-I and IGF-II. ILA pI 4.8 was able to compete, in a parallel manner, with 125I-IGF-I and 125I-IGF-II and, at higher doses, with 125I-insulin in a placental radioreceptor assay. No cross-reactivity was seen in a radioimmunoassay for IGF-I and -II C-peptides, but at higher concentrations parallel displacement was observed in a somatomedin C/IGF-I radioimmunoassay using two different antisera. These data indicate that ILA pI 4.8 does possess many of the biological activities previously reported for the IGFs. Since ILA pI 4.8 does occur naturally in serum, it would appear reasonable to tentatively include it as one of the IGF/somatomedin family.     

Identification of a rabbit liver cytosolic binding protein for human growth hormone.

A specific growth hormone (GH) binding protein of Mr approx. 100000 has been demonstrated in the cytosolic fraction (200000g supernatant) of pregnant-rabbit liver by gel filtration techniques. This binding species was detectable by a standard charcoal separation procedure but not by the widely used poly(ethylene glycol) precipitation method. The GH binding protein had similar binding characteristics to those of classical membrane-bound GH receptors. The kinetics of association and dissociation, binding affinity (2.56 X 10(9)1/mol) and hormonal specificity have been established. There appears to be equal or greater amounts of GH binding protein in the cytosol than in the membrane fraction. The presence of the GH binding protein in rabbit liver cytosol was substantiated by its selective purification on a GH-Affigel 15 affinity column. This technique has resulted in a 200-300-fold purification with no substantial change in binding affinity. The ability of a concanavalin A-Sepharose affinity column to also bind the cytosolic binding protein indicates that, like the membrane-bound GH receptor, it is a glycoprotein. This is the first report of a cytosolic binding protein for GH and raises important questions regarding its potential physiological role in the mechanism of action of GH.     

Ontogenesis of hepatic growth hormone receptors in the rat

Detailed ontogenic studies of the binding of human (hGH) and bovine growth hormone (bGH) have been performed in liver preparations from male and female rats during the neonatal, weanling, pre- and post-pubertal periods. Specific binding of both hormones was readily detected at all ages, with no apparent interference due to occupancy by endogenous hormones. No sex difference in binding was observed prior to weaning (22 days) for hGH, which binds to both somatotrophic and lactogenic sites. However, after weaning a marked sex-related dissociation in the pattern of binding did occur, with female rats binding 3-4 times more hGH than in the pre-weaning period and male rats binding hGH to only half their pre-weaning levels. A very similar pattern was seen for binding of bGH (which binds only to somatotrophic sites) except that in male rats, the post-weaning levels did not fall. Binding patterns for either hGH or bGH prior to weaning did not mirror the known age-related pattern of circulating rat GH levels, suggesting the absence of a definitive auto-regulation system for the GH-GH receptor system under normal circumstances in vivo. The possible role of the weaning process per se in the post-weaning changes of GH binding seen in male and female rats still requires elucidation.     

The heparin binding domain of insulin-like growth factor binding protein (IGFBP)-3 increases susceptibility of IGFBP-3 to proteolysis.

IGFBP-3 proteolysis clears IGFBP-3 from body fluids and increases IGF bioavailability. As shown here, native human IGFBP-3 was cleaved by proteases in media conditioned by hamster and insect cells. This proteolysis was less pronounced for IGFBP-3 containing a mutated heparin binding domain, and was prevented by purifying IGFBP-3 on an IGF-I affinity column in the presence of 2 M sodium chloride, suggesting that the responsible protease(s) binds the IGFBP-3 heparin binding domain. To determine if any human proteases act this way, we first studied plasma prekallikrein since it can copurify with IGFBP-3, and found: 1) [125]IGFBP-3 binds to prekallikrein immobilized either on nitrocellulose or on immunocapture plates; 2) the IGFBP-3 heparin binding domain participates in forming the IGFBP-3/prekallikrein complex; 3) the binary IGFBP-3/prekallikrein complex can bind IGF-I to form a ternary complex; and 4) activation of prekallikrein to alpha-kallikrein by Factor XIIa resulted in proteolysis of bound IGFBP-3. This work suggests: 1) cleavage of IGFBP-3 by a protease may be aided by the ability of the protease zymogen to directly bind the IGFBP-3 heparin binding domain; and 2) direct binding of protease zymogens to IGFBP-3 may explain some instances where IGFBP-3 is preferentially proteolyzed in the presence of other IGFBPs.     

In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

Background

The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.

Results

A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.

Conclusion

In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users.     

Regulation of IFN response gene activity during infliximab treatment in rheumatoid arthritis is associated with clinical response to treatment

Introduction  

Cross-regulation between TNF and type I IFN has been postulated to play an important role in autoimmune diseases. Therefore, we determined the effect of TNF blockade in rheumatoid arthritis (RA) on the type I IFN response gene activity in relation to clinical response.     

Disease-modifying antirheumatic drugs are associated with a reduced risk for cardiovascular disease in patients with rheumatoid arthritis: a case control study

Rheumatoid arthritis (RA) is characterized by inflammation and an increased risk for cardiovascular disease (CVD). This study investigates possible associations between CVD and the use of conventional disease-modifying antirheumatic drugs (DMARDs) in RA. Using a case control design, 613 RA patients (5,649 patient-years) were studied, 72 with CVD and 541 without CVD. Data on RA, CVD and drug treatment were evaluated from time of RA diagnosis up to the first cardiovascular event or the end of the follow-up period. The dataset was categorized according to DMARD use: sulfasalazine (SSZ), hydroxychloroquine (HCQ) or methotrexate (MTX). Odds ratios (ORs) for CVD, corrected for age, gender, smoking and RA duration, were calculated per DMARD group. Patients who never used SSZ, HCQ or MTX were used as a reference group. MTX treatment was associated with a significant CVD risk reduction, with ORs (95% CI): 'MTX only', 0.16 (0.04 to 0.66); 'MTX and SSZ ever', 0.20 (0.08 to 0.51); and 'MTX, SSZ and HCQ ever', 0.20 (0.08 to 0.54). The risk reductions remained significant after additional correction for the presence of rheumatoid factor and erosions. After correction for hypertension, diabetes and hypercholesterolemia, 'MTX or SSZ ever' and 'MTX, SSZ and HCQ ever' showed significant CVD risk reduction. Rheumatoid factor positivity and erosions both increased CVD risk, with ORs of 2.04 (1.02 to 4.07) and 2.36 (0.92 to 6.08), respectively. MTX and, to a lesser extent, SSZ were associated with significantly lower CVD risk compared to RA patients who never used SSZ, HCQ or MTX. We hypothesize that DMARD use, in particular MTX use, results in powerful suppression of inflammation, thereby reducing the development of atherosclerosis and subsequently clinically overt CVD.