Modulation of auxin-binding proteins in cell suspensions

Summary Cultured cell lines from carrot (Daucus carota L.) with little or no embryogenic potential were examined for the auxin-binding capacity of their membranes. The lines belonged to different classes: (a) wild-type lines kept in culture for different periods (the longer the period, the lower being their embryogenic potential); (b) variants, isolated after mutagenesis, showing normal growth but a lack of embryogenic response; (c) auxin-resistant lines, isolated as colonies on solid media containing 45 M 2,4-d; (d) a previously described tumorous line (E9) isolated because of its resistance to hypomethylating drugs. All of these lines showed alterations in auxin-induced, auxin-binding capacity (modulation), i.e. in the non-embryogenic lines the addition of auxin increased the auxinbinding capacity to a very small degree, or removal of the hormone did not produce the proper decrease in that capacity, or both defects could be simultaneously present. Both types of defects were shown to be correctable: after treatments designed to increase the amplitude of modulation, embryogenic capacity was restored in a number of lines.     

Gly-238-Ser substitution changes the substrate specificity of the SHV class A beta-lactamases

The SHV-type beta-lactamase SHV-2A is related to SHV-1 by a Gly-238-Ser replacement. Strains carrying SHV-2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV-1 are sensitive to these drugs. We present a kinetic analysis of a SHV-1 and SHV-2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV-2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7-aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV-1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV-2A. These results indicate that the Ser-238-Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV-2A relative to SHV-1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser-238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV-2A. We propose that the Gly-238 to Ser-238 replacement in SHV-2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.     

A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn3 mutant

This paper describes a simple method for the isolation of small plasmids of various sizes from pSMI, a derivative of the resistance plasmid R 100. The method is based on the observation that a repressor-negative mutant of the ampicillin-resistance (amp^r) transposon Tn3, Tn3 No. 5, mediates cointegration of a plasmid carrying Tn 3 No. 5 (pMB8::Tn 3 No. 5) into virtually any site on pSMI. The resulting cointegrate plasmids contain the pSMI sequence which is joined with the amp^r gene of the Tn 3 mutant. This cointegration is so frequent that large cointegrate plasmids can be readily detected in the total plasmid DNA prepared from cells carrying pSMI and pMB8::Tn3 No. 5. We were able to isolate small plasmids of various sizes by digesting the total plasmid DNAs with restriction endonucleases which cut both pSM 1 and Tn3 No. 5 sequences present in the cointegrates and subsequently ligating the restriction fragment containing both the amp^r gene and the region necessary for replication of pSMI. Analysis of these plasmids, named pBV plasmids, with restriction endonucleases and by nucleotide sequencing allowed us to determine regions necessary or unnecessary for replication, thus defining a minimal replication region of pSMI. The present method is generally useful for the isolation of small derivatives from any large plasmid for the study of genes and sites adjacent to or within the minimal replication region of the plasmid.     

Leishmania tarentolae: streptomycin and chloramphenicol resistance of promastigotes.

Stable mutant strains of Leishmania tarentolae promastigotes resistant to chloramphenicol (CAP) were isolated by replica-plating techniques. In addition, cell lines stress-adapted to streptomycin and to high culture temperature (33 C) were obtained. Drug resistance was influenced by temperature, culture media, and plating technique. Inhibition of amino acid incorporation into protein occurs in CAP-resistant cells when exposed to 600 μg CAP/ml but this inhibition was 50–80% lower than that found in wild type sensitive cells. The primary site of CAP action appears to be inhibition of protein synthesis. CAP also adversely affected proline oxidation.     

Two plant NLR proteins confer strain-specific resistance conditioned by an effector from Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae(Psa) causes bacterial canker, a devastating disease threatening the Actinidia fruit industry. In a search for non-host resistance genes against Psa, we find that the nucleotidebinding leucine-rich repeat receptor(NLR) protein ZAR1 from both Arabidopsis and Nicotiana benthamiana(Nb) recognizes Hop Z5 and triggers cell death. The recognition requires ZED1 in Arabidopsis and JIM2 in Nb plants, which are members of the ZRK pseudokinases and known components of the...     

Cucurbit Powdery Mildew: First Insights for the Identification of the Causal Agent and Screening for Resistance of Squash Genotypes (Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir.) in Mendoza,Argentina

The cucurbit powdery mildew (CPM) caused by different fungal species is a major concern for cucurbit crops around the world. In Argentina CPM constitutes the most common and damaging disease for cucurbits, especially for squash crops (Cucurbita moschata). The present study displays initial insights into the knowledge of the disease in western Argentina, including the determination of the prevalent species causing CPM, as well as the evaluation of the resistance of squash cultivars and breeding lines. Fungal colonies were isolated from samples collected in Mendoza province, Argentina. A field trial was also performed to assess the resistance of five squash accessions, including commercial cultivars and breeding lines. The severity of CPM was analyzed and epidemiological models were built based on empirical data. The morphological determinations and analysis with specific molecular markers confirmed Podosphaera xanthi as the prevalent causal agent of CPM in Mendoza. The results od the field trial showed differences in the resistance trait among the squash accessions. The advanced breeding line BL717/1 showed promising results as source of CPM resistance for the future development of open pollinated resistant cultivars, a crucial tool for an integrative control of the disease.     

Mitotracker Green Is a P-Glycoprotein Substrate

P-glycoprotein has a widespread expression on normal tissues. The protein has also been strongly associated with the multidrug resistance phenotype (MDR) on tumor cells. The employment of flow cytometry and confocal microscopy has contributed to the discovery and application of new particular fluorescent dyes. Nevertheless, several studies are being performed in different cellular types neglecting the expression/activity of MDR proteins. Because many fluorochromes have been reported as P-glycoprotein substrates, an especial attention must be given to the properties of new dyes in the presence of MDR proteins. Flow cytometric analyzes of Mitotracker Green (MTG) fluorescence profile were performed in a human erythroleukemic cell line and its resistant counterpart. In this report we demonstrated that MTG, a probe used to evaluate the mitochondrial mass, is a P-glycoprotein substrate and its staining profile is dependent on the activity of this protein. In vitro studies on a human erythroleukemic cell line and its resistant counterpart revealed that MDR modulators (Cyclosporin A, Verapamil, and Trifluoperazine) alter the MTG fluorescence pattern on a resistant cell line. The findings suggest that attention should be given to the expression of P-glycoprotein when performing an evaluation of mitochondria properties with MTG.     

Probiotics as an emerging therapeutic strategy to treat NAFLD: focus on molecular and biochemical mechanisms

Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, both in adults and in children. NAFLD is characterized by aberrant lipid storage in hepatocytes (hepatic steatosis) and inflammatory progression to nonalcoholic steatohepatitis. Evidences so far suggest that intrahepatic lipid accumulation does not always derive from obesity. Gut microbiota has been considered as a regulator of energy homeostasis and ectopic fat deposition, suggesting its implications in metabolic diseases. Probiotics are live microbial that alter the enteric microflora and have beneficial effects on human health. Although the molecular mechanisms of probiotics have not been completely elucidated yet, many of their effects have proved to be beneficial in NAFLD, including the modulation of the intestinal microbiota, an antibacterial substance production, an improved epithelial barrier function and a reduced intestinal inflammation. Given the close anatomical and functional correlation between the bowel and the liver, and the immunoregulatory effects elicited by probiotics, the aim of this review is to summarize today's knowledge about probiotics in NAFLD, focusing in particular on their molecular and biochemical mechanisms, as well as highlighting their efficacy as an emerging therapeutic strategy to treat this condition.     

Assessment of variability among morphological and molecular characters in wild populations of mint [Mentha longifolia (L.) L.] germplasm

Mentha longifolia is an important medicinal and aromatic perennial herb that exhibits wide distribution range from sub-tropical to temperate regions. In the present study, agro-morphological traits and genetic differences in 19 different populations of M. longifolia were studied to evaluate the level and extent of its diversity. Analysis of variance (ANOVA) showed that the different phenotypic characters show considerable differences among various populations and was significant at p < 0.05. Molecular diversity analysis performed by using arbitrary amplified eleven ISSR primers generated a total of 121 amplicons that range within the size of 200–2500 base pairs (bp). Each primer on average generated 11 amplicons with percentage polymorphism being 100. The analysis of molecular variance (AMOVA) showed more (64%) among population genetic diversity and less (36%) within the populations. Greater genetic differentiation (Gst = 0.6852) among these populations occurs due to low gene flow (Nm = 0.2297) and greater habitat variability. Geographic and genetic distances were positively correlated according to Mantel’s test. In order to remove any kind of biases, we used R software to perform cluster and redundancy analysis to analyse the extent of relatedness among studied populations. In terms of morphological and molecular aspects, the populations were grouped into four and five clusters respectively based on hierarchical clustering method. The results demonstrated that M. longifolia displays a great degree of morphological and genetic variation and can be utilized in breeding, genetic improvement, and gene bank conservation programmes in future.     

Specific and Selective Bacteriophages in the Fight against Multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii causes serious infections especially in immunocompromised and/or hospitalized patients. Several A. baumannii strains are multidrug resistant and infect wounds, bones, and the respiratory tract. Current studies are focused on finding new effective agents against A. baumannii. Phage therapy is a promising means to fight this bacterium and many studies on procuring and applying new phages against A. baumannii are currently being conducted. As shown in animal models, phages against multidrug-resistant A. baumannii may control bacterial infections caused by this pathogen and may be a real hope to solve this dangerous health problem.