First Report of Root-Knot Nematode Meloidogyne javanica on Chrysanthemum in Colombia.

Severe plant stunting, chlorosis, and extensive root galling were observed on chrysanthemum (Dendrathema grandiflora cv. Yellow Vero) in a commercial cut-flower production facility in Rionegro, Antioquia, northwestern Colombia. Examination of the root samples from selected infected plants revealed the presence of abundant root-knot nematodes. Juveniles, males, and females were extracted for species identification using morphological characteristics. Identification was confirmed by perineal patterns and esterase phenotype analysis of females. All methods of identification were consistent with typical Meloidogyne javanica. No other root-knot nematode species were found on this farm, but the presence of other Meloidogyne species in the region is possible. Root-knot nematodes have been reported to cause economic losses in cut-flower plantations in Colombia (1), but there are no reports of the species involved. M. javanica has an extensive host range and wide distribution. The identification and distribution of M. javanica in chrysanthemum production is relevant because nematode-fungus interactions may depend on the nematode species involved. Only M. javanica, and not M. hapla or M. incognita, has been found to increase the severity of Fusarium wilt on chrysanthemum (2). To our knowledge, this is the first report of M. javanica on chrysanthemum in Colombia. References: (1) G. Arbeláez. Acta Hortic. 482:91, 1999. (2) A. W. Johnson and R. H. Littrell. J. Nematol. 1:122. 1969.

Evaluación de la sinceridad del esfuerzo en el hombro mediante dinamometría isocinética / Evaluation of the sincerity of the efforts in the shoulder by isokinetic dynamometry

La determinación del déficit de fuerza muscular como secuela es fundamental en la rehabilitación laboral. Para definir un déficit muscular se requiere una colaboración máxima del paciente en la realización del esfuerzo. La valoración de la colaboración de los pacientes en la realización de las pruebas dinamométricas se ha venido realizando mediante el coeficiente de variación, aunque la literatura reciente cuestiona esta aproximación y propone otras basadas en la fisiología de la contracción muscular máxima en modalidades concéntrica y excéntrica. El objetivo de este trabajo es analizar nuestra experiencia en la evaluación de la fuerza y la sinceridad del esfuerzo de la musculatura del hombro mediante dinamometría isocinética. Material y métodos: Se aplica el protocolo evaluador a 14 pacientes (13 varones y una mujer; edad media 46 años; la mayoría trabajadores manuales) remitidos a la unidad de isocinesia de una mutua laboral para peritación de la fuerza muscular del hombro. La patología más frecuente fue la ruptura de manguito intervenida. El protocolo comprende la determinación de los momentos de fuerza máximos de ambos hombros en rotaciones, abducción y flexoextensión a diferentes velocidades (60º/s, 120º/s y 180º/s) y dos modalidades de contracción (concéntrica y excéntrica). A continuación, se calculan los déficit de fuerza y los ratios contracción excéntrica/concéntrica (REC).Un déficit mayor al 20 por ciento se considera relevante y un REC superior al 2,05 sugestivo de poca colaboración. Resultados: Doce pacientes mostraron déficit relevantes de fuerza muscular, de los cuales cinco presentaron indicios de poca colaboración. Tres aceptaron la propuesta de alta basada en los resultados, uno continua de baja y en un caso fue utilizada como prueba en magistratura para desestimar la petición de incapacidad del paciente. Discusión: La medición de la fuerza muscular mediante dinamometría isocinética debería acompañarse siempre de una valoración del grado de colaboración del paciente. La aproximación propuesta en este trabajo nos ha sido útil en un entorno laboral (AU)

The native state conformational ensemble of the SH3 domain from alpha-spectrin.

The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the beta-strands, the short 3(10) helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the beta-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53-57) and a highly unstable region, the loop between residues 34-41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global.

Physical organization of the upper pathway operon promoter of the Pseudomonas TOL plasmid. Sequence and positional requirements for XylR-dependent activation of transcription.

The upper pathway operon of the Pseudomonas putida TOL plasmid belongs to the -12/-24 class of promoters. These promoters exhibit three regions critical for regulated transcription, namely, the -12/-24 site for RNA polymerase/sigma 54 binding, the -55/-67 region for IHF protein binding, and the -130(UAS2)/-170(UAS1) region, where two sites for XylR binding are located. The XylR-protected G residues located at -131, -139, -160 and -169 were replaced with As, and the activity of the mutant promoters was assayed after fusion to a promoterless lacZ gene. The mutation (G(-169)-->A) resulted in a 50% decrease in expression from the promoter (Pu), whereas the other three changes had no significant effect. The XylR recognition sequence UAS2 has a perfect inverted repeat (5'-ATTTN4-AAAT-3') while UAS1 shows two mismatches (5'-CCTTN4AAAT-3'). The two Cs (located at -172 and -173), which interrupt the inverted repeat, were changed as follows: C(-172)-->T; C(-173)-->A, CC(-172, -173)-->AT. Transcription activation from the mutant promoters was measured as beta-galactosidase activity after fusion to lacZ; the better the palindromic sequence, the higher the rate of transcription from Pu, with increases in activity of up to 50%. The introduction of one or two full helix turns between the IHF and the XylR binding sites did not significantly affect transcription from Pu; however, the insertion of three helix turns resulted in a drop of 90% in the activity. The non-permissive effect of insertion of three full helix turns between the IHF and XylR binding sites was not evident in an IHF- background.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitroaromatics Are Substrates for the TOL Plasmid Upper-Pathway Enzymes.

Expression of the xylMA genes encoding for toluene monoxygenase from the lactose promoter in a broad-host-range plasmid allows the oxidation of toluene and m- and p-nitrotoluene to their corresponding benzyl alcohols and benzaldehydes in Pseudomonas putida and Escherichia coli. Benzyl alcohols accumulate until reaching a concentration of about 80 muM, while benzaldehydes accumulate steadily with time for at least 24 h. TOL-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase recognize m- and p-nitro-substituted compounds as substrates. In contrast, the XylR protein, which regulates the TOL plasmid-encoded upper-pathway operon, does not recognize nitro-substituted toluenes as effectors.

Activation of the Pseudomonas TOL plasmid upper pathway operon. Identification of binding sites for the positive regulator XylR and for integration host factor protein.

Expression of the Pseudomonas putida TOL plasmid upper pathway operon requires a promoter that belongs to the -12/-24 class. Stimulation of transcription from this promoter is positively controlled by the effector-activated XylR protein and requires a form of RNA-polymerase holoenzyme containing the RpoN-encoded sigma factor, sigma 54. XylR-dependent stimulation of transcription from the Pseudomonas TOL upper pathway promoter was examined using deletions, insertions, and in vivo dimethyl sulfate footprinting. Two upstream activator sequences were identified in the -160 (UAS1) and -130 (UAS2) regions. Deletion of these two regions abolished transcription activation, although conservation of the UAS2 element alone allowed limited transcription stimulation. Separation of UAS1 from UAS2 by half a turn or a full turn significantly reduced XylR stimulation of transcription from the upper pathway operon promoter. An inverted repeated ATTTGN2CAAAT (where N is any nucleoside), which most likely represented the XylR recognition sequence, was identified. Binding of XylR was observed in vivo in the absence of effector, but changes in the binding pattern were induced in the presence of m-methylbenzyl alcohol, a XylR effector. In vivo footprinting analysis revealed that changes in the methylation pattern of G and T also occurred in the -50 to -90 region, which is probably occupied by integration host factor (IHF) protein. IHF was required for maximal expression from the TOL upper pathway operon promoter in Escherichia coli. Separation of the IHF site from UAS2 by a full helix turn did not significantly affect stimulation of transcription, which is consistent with this region playing a conformational role, rather than a regulatory one, in promoter function.

Promoter-upstream activator sequences are required for expression of the xylS gene and upper-pathway operon on the Pseudomonas TOL plasmid.

Stimulation of transcription from the Pseudomonas TOL plasmid xylS gene promoter (Ps) and the upper-pathway operon promoter (Pu) is dependent on the positive regulator protein XylR activated by an effector molecule such as 3-cholorotoluene, and on RpoN, an RNA polymerase sigma factor. Mutational analysis of the Ps and Pu promoters showed that upstream activator sequences located between -110 and -218bp upstream of the main transcription initiation point are required for regulated expression from these promoters. A search for homologous nucleotide sequences in the -110 to -218bp region in Pu and Ps revealed conserved sequences that may act as putative recognition sequences for the XylR protein. Ps and Pu exhibit another well-conserved region at around -50bp, which is homologous to corresponding sites in other RpoN-dependent promoters and may constitute a binding site for integration host factor (IHF).

Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway.

The TOL plasmid upper pathway operon encodes enzymes involved in the catabolism of aromatic hydrocarbons such as toluene and xylenes. The regulator of the gene pathway, the XylR protein, exhibits a very broad effector specificity, being able to recognize as effectors not only pathway substrates but also a wide variety of mono- and disubstituted methyl-, ethyl-, and chlorotoluenes, benzyl alcohols, and p-chlorobenzaldehyde. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two upper pathway enzymes, exhibit very broad substrate specificities and transform unsubstituted substrates and m- and p-methyl-, m- and p-ethyl-, and m- and p-chloro-substituted benzyl alcohols and benzaldehydes, respectively, at a high rate. In contrast, toluene oxidase only oxidizes toluene, m- and p-xylene, m-ethyltoluene, and 1,2,4-trimethylbenzene [corrected], also at a high rate. A biological test showed that toluene oxidase attacks m- and p-chlorotoluene, albeit at a low rate. No evidence for the transformation of p-ethyltoluene by toluene oxidase has been found. Hence, toluene oxidase acts as the bottleneck step for the catabolism of p-ethyl- and m- and p-chlorotoluene through the TOL upper pathway. A mutant toluene oxidase able to transform p-ethyltoluene was isolated, and a mutant strain capable of fully degrading p-ethyltoluene was constructed with a modified TOL plasmid meta-cleavage pathway able to mineralize p-ethylbenzoate. By transfer of a TOL plasmid into Pseudomonas sp. strain B13, a clone able to slowly degrade m-chlorotoluene was also obtained.