Efficiency of IRAP and ITS-RFLP marker systems in accessing genetic variation of Pyrenophora graminea

The usefulness of IRAP (inter-retrotransposon amplified polymorphism) and ITS-RFLP (restriction of PCR-amplified internal transcribed spacers of the rDNA) markers in the analysis of 39 Pyrenophora graminea isolates was determined. Each marker system could discriminate between all of the isolates in detecting polymorphism, albeit with variable efficiency. IRAP and ITS-RFLP produced 85% and 77% polymorphic bands, respectively, with a corresponding mean polymorphic information content (PIC) of 0.38 and 0.36. The IRAP marker index ratio (2.41) was higher than ITS-RFLP (1.50). On one hand, the quality nature of data (QND) was higher for ITS-RFLP (0.169) than IRAP (0.093). However, correlation between both marker similarity matrices was significant (r = 0.34, p < 0.05). These findings suggest their combined use in phylogenetic analysis. To our knowledge, this is the first report of a comparison involving these two advanced DNA marker systems.     

Glycosylation of Pilin and Nonpilin Protein Constructs by Pseudomonas aeruginosa 1244

PilO is an oligosaccharyl transferase (OTase) that catalyzes the O-glycosylation of Pseudomonas aeruginosa 1244 pilin by adding a single O-antigen repeating unit to the β carbon of the C-terminal residue (a serine). While PilO has an absolute requirement for Ser/Thr at this position, it is unclear if this enzyme must recognize other pilin features. To test this, pilin constructs containing peptide extensions terminating with serine were tested for the ability to support glycosylation. It was found that a 15-residue peptide, which had been modeled on the C-proximal region of strain 1244 pilin, served as a PilO substrate when it was expressed on either group II or group III pilins. In addition, adding a 3-residue extension culminating in serine to the C terminus of a group III pilin supported PilO activity. A protein fusion composed of strain 1244 pilin linked at its C terminus with Escherichia coli alkaline phosphatase (which, in turn, contained the above-mentioned 15 amino acids at its C terminus) was glycosylated by PilO. E. coli alkaline phosphatase lacking the pilin membrane anchor and containing the 15-residue peptide was also glycosylated by PilO. Addition of the 3-residue extension did not allow glycosylation of either of these constructs. Site-directed mutagenesis of strain 1244 pilin residues of the C-proximal region common to the group I proteins showed that this structure was not required for glycosylation. These experiments indicate that pilin common sequence is not required for glycosylation and show that nonpilin protein can be engineered to be a PilO substrate.Colonization and dissemination of the opportunistic pathogen Pseudomonas aeruginosa rely to a large extent on the ability of this organism to produce functional type IV pili (26). These protein fibers, which radiate from the cell pole, are adhesion factors (51), mediate a form of surface translocation referred to as twitching motility (10, 37), and are important in biofilm formation (39). The pili of this organism are primarily composed of a monomeric subunit called pilin (PilA). Type IV pili can be differentiated into two classes (a or b) on the basis of the PilA sequence and structure (23). Although they display considerable sequence variation, the majority of the type IVa pilins of P. aeruginosa can be placed into one of three groups on the basis of primary structure and antigenicity, as well as by the presence of auxiliary pilin genes found immediately downstream from pilA (8, 33). We previously determined that pilin from P. aeruginosa 1244, which belongs to group I (8), contained an O-antigen repeating unit covalently attached to the β-hydroxyl group of a serine residing at the C terminus of this protein (7). While the specific physiological role of the pilin glycan in this organism is not clear, the presence of this saccharide influences pilus hydrophobicity and has a pronounced effect on virulence, as determined in a mouse respiratory model (47). The metabolic origin of the pilin saccharide is the O-antigen biosynthetic pathway (14), and its attachment is catalyzed by an oligosaccharyl transferase (OTase) called PilO (6). Specific regions of this cytoplasmic membrane protein necessary for glycosylation activity have been identified (42). Topological studies of PilO have shown that these regions face the periplasm, suggesting that pilin glycosylation takes place in this chamber (42). Here the glycan substrate is the O-antigen repeating unit covalently linked to the undecaprenol carrier lipid.PilO has a very relaxed glycan substrate specificity, as indicated by the evidence that it is able to utilize a number of structurally dissimilar O-antigen repeating units as substrate (14), and requires only features of the reducing end sugar to carry out pilin glycosylation (28). WaaL, the enzyme that transfers polymerized O antigen to core lipid A, from Escherichia coli also has a similar broad glycan specificity (19). Recent studies (18) provided evidence that PglL, an OTase of Neisseria meningitidis, recognized only the carrier lipid and was able to attach a variety of saccharides to the pilin of this organism. Although the glycan specificity of PilO is relaxed, this enzyme will not attach other carrier lipid-bound saccharides, such as the peptidoglycan subunit or polymerized O-antigen repeating unit, to pilin. This is indicated by the absence of pilins with increased mass in O-antigen-negative mutants or the production of multiple pilin sizes in the wild-type strain (6).In vivo analysis of mutagenized P. aeruginosa 1244 pilin showed that the C-terminal serine of this protein was a major pilin glycosylation recognition feature of PilO (27). In addition, modification (substitution of the C-terminal amino acid with a 3-residue sequence terminating in serine) of a group II pilin allowed PilO-dependent attachment of the O-antigen repeating unit (27). While these results suggested that the preponderance of pilin structural information was not required for glycosylation, it was not clear whether regions common among the P. aeruginosa pilins were needed. In the present study three types of experiments were carried out in order to answer this question. First, the glycosylation site was extended away from the pilin surface with the addition of a 15-residue peptide which terminates with serine. Second, an engineered periplasmic protein containing the glycosylation residue at its C terminus was fused with pilin and tested for PilO activity. Finally, this periplasmic protein containing no pilin common region was constructed and tested. Evidence presented in this paper suggests that PilO requires only the glycosylation target residue.The work presented also indicated that, in addition to pilins, nonpilin protein free in the periplasm or anchored to the cytoplasmic membrane could be engineered so as to serve as a PilO substrate. These results suggest that a wide range of pilins and nonpilin proteins can be engineered to serve as substrate for glycosylation, a finding that would potentially have practical value, particularly in the area of vaccine construction. In addition to elucidating the protein specificity of the PilO system, the present work determined that the peptide extension used can supply functional epitope information to the modified protein, in addition to providing a site for glycosylation. Altogether, the results presented suggest that engineering of pilins and nonpilin proteins for the biological generation of protein-peptide-saccharide constructs is a potentially important strategy in vaccine design.     

Rho-ROCK-Myosin Signaling Meditates Membrane Type 1 Matrix Metalloproteinase-induced Cellular Aggregation of Keratinocytes

Membrane type 1-matrix metalloproteinase (MT1-MMP, MMP14), which is associated with extracellular matrix (ECM) breakdown in squamous cell carcinoma (SCC), promotes tumor formation and epithelial-mesenchymal transition. However, in this report we demonstrate that MT1-MMP, by cleaving the underlying ECM, causes cellular aggregation of keratinocytes and SCC cells. Treatment with an MMP inhibitor abrogated MT1-MMP-induced phenotypic changes, but decreasing E-cadherin expression did not affect MT1-MMP-induced cellular aggregation. As ROCK1/2 can regulate cell-cell and cell-ECM interaction, we examined its role in mediating MT1-MMP-induced phenotypic changes. Blocking ROCK1/2 expression or activity abrogated the cellular aggregation resulting from MT1-MMP expression. Additionally, blocking Rho and non-muscle myosin attenuated MT1-MMP-induced phenotypic changes. Moreover, SCC cells expressing only the catalytically active MT1-MMP protein demonstrated increased cellular aggregation and increased myosin II activity in vivo when injected subcutaneously into nude mice. Together, these results demonstrate that expression of MT1-MMP may be anti-tumorigenic in keratinocytes by promoting cellular aggregation.     

A procedure for the purification of beta-lactoglobulin from bovine milk using gel filtration chromatography at low pH

A simple and rapid procedure for the purification of beta-lactoglobulin (β-LG) from bovine milk is described. The procedure exploits the major difference in molecular mass of β-LG and other whey components and the existence of the former in monomeric form at acidic pH. Gel filtration of whey was carried out using a Bio-Gel P10 column at pH 3.0. Residual caseins and other milk proteins were excluded from the gel and β-LG and alpha-lactalbumin (α-LA) emerged as two fully resolved peaks. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that β-LG was purified to apparent homogeneity, while absorption, fluorescence, and circular dichroism spectroscopy indicated the native-like conformation of the protein. Western blot analysis revealed that the antibodies raised against the purified β-LG in rabbits also readily react with the commercial bovine protein. This procedure requires only 4-5 hr for the purification of about 10 mg of β-LG from a single run while using a small column (2.3 cm x 83 cm) of Bio-Gel P10 and has the potential for scaling up.     

Sequence analysis of the gene encoding H antigen in <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from food in Morocco

In order to develop other molecular method useful for typing of motile and non motile Escherichia coli strains, a total of 207 strains of E. coli (133 reference strains, 74 food strains) were characterized by analysis of sequences of their amplified flagellin-encoding (fliC) gene products. The collection of reference strains was used for database building of fliC gene sequences. Application of this identification system to 74 E. coli food isolates revealed a reproducible and clear cut classification with very good correlation to results obtained by HhaI restriction of the amplified flagellin gene. The proposed determination of fliC sequences variations should be helpful for epidemiological studies.     

Effect of grain type and processing method on rumen fermentation and milk rumenic acid production

It was hypothesized that differences in starch degradability account for observed differences in rumen vaccenic acid (t11-18:1) and milk rumenic acid (RA) concentrations. To test this hypothesis, starch degradability was varied through grain source and by processing. Eight Holstein cows in mid-lactation were assigned to two 4 × 4 Latin squares with four 21-day periods and four diets: dry rolled barley, ground barley, dry rolled corn and ground corn. Diets contained similar starch content and were supplemented with whole sunflower seed to provide similar total polyunsaturated fatty acid (PUFA) (18:2n-6 + 18:3n-3) contents. Forage/concentrate ratios of all diets were 42 : 58. Rumen, plasma and milk samples were collected in the third week of each period. In situ degradation rates (%/h) for rolled corn, ground corn, rolled barley and ground barley were 5.4, 8.9, 17.0 and 19.4, respectively, for dry matter (DM) and 6.3, 10.8, 25.3 and 43.8, respectively, for starch. DM intakes were greater for corn-based diets (CBD) than for barley-based diets (BBD) with no difference between rolled and ground diets. Daily minimum rumen pH was less (5.2 v. 5.5) and pH duration <5.8 (h/d) was greater (7.4 v. 4.3) for BBD than for CBD. Milk fat content and yield were less for BBD than for CBD with greater values observed for rolling compared with grinding. Variability in milk fat yield was strongly related (R2 = 0.55; P < 0.01) to total starch intake (45%) and milk c9t11-CLA (10%) and none of the t-18:1 isomers or CLA isomers that are typically associated with milk fat depression entered the model. The concentrations (%) of t10-18:1 and t11-18:1 were greater for BBD than for CBD in rumen contents (t10-18:1, 3.5 v. 1.3; t11-18:1, 3.2 v. 1.9), plasma (t10-18:1, 1.2 v. 0.2; t11-18:1, 0.97 v. 0.58) and milk (t10-18:1, 3.8 v. 1.0; t11-18:1, 2.6 v. 1.7) despite greater total PUFA intakes for CBD. Milk RA concentration was greater for BBD than for CBD (1.46 v. 0.89) but was not influenced by the method of grain processing. This study clearly demonstrated that the milk content and profile of t-18:1 and CLA isomers were more strongly influenced by the source of grain starch (barley > corn) than by the method of grain processing indicating that factors inherent in the source of starch were responsible for the observed differences and these factors could not be modified by the processing methods used in this study.     

A molecular mechanism for eflornithine resistance in African trypanosomes

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.     

Development of a cultivation-independent approach for the study of genetic diversity of Sinorhizobium meliloti populations

The development of a species-specific marker for the analysis of the genetic polymorphism of the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti directly from environmental DNA is reported. The marker is based on terminal-restriction fragment length polymorphism (T-RFLP) methodology targeting specifically the 16S-23S Ribosomal Intergenic Spacer of S. meliloti. Species-specificity and polymorphism of the marker were tested on DNA extracted from soil samples and from a collection of 130 S. meliloti bacterial isolates. These primers and the T-RFLP approach proved useful for the detection and analysis of polymorphism of S. meliloti populations.     

Neuroanatomical study of the A11 diencephalospinal pathway in the non-human primate

Background

The A11 diencephalospinal pathway is crucial for sensorimotor integration and pain control at the spinal cord level. When disrupted, it is thought to be involved in numerous painful conditions such as restless legs syndrome and migraine. Its anatomical organization, however, remains largely unknown in the non-human primate (NHP). We therefore characterized the anatomy of this pathway in the NHP.

Methods and Findings

In situ hybridization of spinal dopamine receptors showed that D1 receptor mRNA is absent while D2 and D5 receptor mRNAs are mainly expressed in the dorsal horn and D3 receptor mRNA in both the dorsal and ventral horns. Unilateral injections of the retrograde tracer Fluoro-Gold (FG) into the cervical spinal enlargement labeled A11 hypothalamic neurons quasi-exclusively among dopamine areas. Detailed immunohistochemical analysis suggested that these FG-labeled A11 neurons are tyrosine hydroxylase-positive but dopa-decarboxylase and dopamine transporter-negative, suggestive of a L-DOPAergic nucleus. Stereological cell count of A11 neurons revealed that this group is composed by 4002±501 neurons per side. A 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication with subsequent development of a parkinsonian syndrome produced a 50% neuronal cell loss in the A11 group.

Conclusion

The diencephalic A11 area could be the major source of L-DOPA in the NHP spinal cord, where it may play a role in the modulation of sensorimotor integration through D2 and D3 receptors either directly or indirectly via dopamine formation in spinal dopa-decarboxylase-positives cells.     

β2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.