Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.

Characterization of an oxidative stress response regulator, homologous to Escherichia coli OxyR, from the phytopathogen Xylella fastidiosa.

The OxyR oxidative stress transcriptional regulator is a DNA-binding protein that belongs to the LysR-type transcriptional regulators (LTTR) family. It has the ability to sense oxidative species inside the cell and to trigger the cell's response, activating the transcription of genes involved in scavenging oxidative species. In the present study, we have overexpressed, purified and characterized the predicted OxyR homologue (orf xf1273) of the phytopathogen Xylella fastidiosa. This bacterium is the causal agent of citrus variegated chlorosis (CVC) disease caused by the 9a5c strain, resulting in economic and social losses. The secondary structure of the recombinant protein was analyzed by circular dichroism. Gel filtration showed that XfoxyR is a dimer in solution. Gel shift assays indicated that it does bind to its own predicted promoter under in vitro conditions. However, considering our control experiment we cannot state that this interaction occurs in vivo. Functional complementation assays indicated that xfoxyR is able to restore the oxidative stress response in an oxyr knockout Escherichia coli strain. These results show that the predicted orfxf1273 codes for a transcriptional regulator, homologous to E. coli OxyR, involved in the oxidative stress response. This may be important for X. fastidiosa to overcome the defense mechanisms of its host during the infection and colonization processes.

Overexpression and purification of PWL2D, a mutant of the effector protein PWL2 from Magnaporthe grisea.

The rice blast disease caused by the ascomycete Magnaporthe grisea continues to cause a tremendous impact in rice (Oryza sativa) cultures around the world. Elucidating the molecular basis of the fungus interactions with its host might help increase the general understanding of the pathogen-host relationship. At the moment of invasion, the fungus secretes effectors that modify host defenses and cellular processes as they successively invade living rice cells. PWL2, an effector protein, is a known AVR (avirulence) gene product. The PWL2 gene prevents the fungus from infecting weeping lovegrass (Eragrostis curvula). In this study, we identified a PWL2 allele gene (which we termed PWL2D) in a strain of M. grisea. The sequence of PWL2D has only two bases different from that of PWL2, producing alterations in residue 90 and residue 142. However, the alteration of residue 90 (from D(90) to N(90)) is critical to gene function. Here, we cloned the gene PWL2D in a pET System vector, expressed the gene product in Escherichia coli and evaluated by spectroscopic techniques some aspects of the PWL2D structure. While TRX-tagged PWL2D is prone to aggregation, the solubility of PWL2D is improved when it is overexpressed without its original signal peptide. Expression and purification procedures for these constructs are described. Finally, we found out that the protein seems to be an intrinsically disordered protein. Results from these studies will facilitate structural analysis of PWL2D and might contribute to understanding the gene's function and of fungal/plant interactions.

Gastrin antibodies: induction, demonstration, and specificity.

Antibodies to the antral hormone gastrin have been induced in the rabbit and detected by passive hemagglutination. Specificity of the antibody, as determined by three methods, is directed to gastrins I and II, gastrin pentapeptide, and gastrin tetrapeptide, as well as to the stage-1 gastrin used for immunization.

Immunoblastic sarcoma of T- and B-cell types: morphologic description and comparison.

Immunoblastic sarcoma (IBS) is a large cell lymphoma conceptually related to transformed T and B lymphocytes of the extrafollicular compartment of the immune system (immunoblasts). This light microscopic study of a series of 47 immunologically defined cases of IBS was undertaken in an attempt to define more precisely the morphologic features of the T- and B-cell subtypes. A remarkable morphologic spectrum characterized T-IBS (31 cases), which could be divided into two main groups: 1) tumors composed of varying mixtures of small, medium-sized, and large transformed cells; and 2) tumors with more homogeneous populations of medium-sized or large transformed cells. These cells, in all sizes, generally had abundant pale-staining cytoplasm, delicate nuclear membranes, finely dispersed chromatin, and one to several, small or medium-sized, prominent nucleoli. A distinctive background of small, irregular lymphocytes was frequently present. Plasmacytoid differentiation, seen most consistently as amphophilic staining of the cytoplasm, generally characterized B-IBS (16 cases). B-IBS similarly showed a morphologic spectrum that occurred in two main forms: 1) tumors consisting of a spectrum of transformed cells, with the smaller cells often showing the most striking plasmacytoid differentiation; and 2) tumors consisting predominantly of medium-sized to large transformed cells with varying degrees of plasmacytoid differentiation. With this constellation of features, all but two cases of T-IBS and one case of B-IBS were morphologically distinguishable.

Myelodysplasia in the acquired immune deficiency syndrome.

Certain new hematologic findings in eight homosexual or bisexual patients with the acquired immune deficiency syndrome (AIDS) are presented. All eight patients manifested a normochromic, normocytic anemia, and six of eight had granulocytopenia during their hospitalization. The other two had low-normal granulocyte counts. Bone marrow examination showed normocellular or hypercellular marrows with increased myeloid: erythroid ratios and increased numbers of megakaryocytes. All patients had abnormalities in maturation of all cell lines, most prominent in the granulocytic series. This constellation of features is similar to the findings in the myelodysplastic syndromes (preleukemia). The authors suggest that myelodysplasia in patients with AIDS results in ineffective hematopoiesis and contributes to the peripheral blood cytopenias found in these patients. Myelodysplasia could be a direct or indirect effect of human T-lymphotropic retrovirus-III (HTLV-III).

Cyclic nucleotides in the rat neostriatum: push-pull perfusion studies.

A push-pull perfusion technique was employed for in vivo study of adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) in the rat caudate nucleus. Addition of dopamine to the perfusion fluid elicited dose-dependent increases of both cAMP and cGMP perfusate concentrations. In separate experiments, it was found that pretreatment of animals with the dopamine antagonist, pimozide, significantly depressed both nucleotide responses to dopamine perfusion over the dose range studied. Mechanistic interpretations of the observations are considered. The push-pull perfusion technique appears to provide an extremely useful means of examining extracellular cyclic nucleotide levels in a discrete brain region, in vivo, under dynamic conditions.

Ethanol effects on harmaline-induced tremor and increase of cerebellar cyclic GMP.

The spectra of pharmacological effects of ethanol and the benzodiazepine show a degree of overlap. Neurophysiological and neurochemical evidence indicates that both ethanol and benzodiazepines facilitate inhibitory neurotransmission mediated by GABA. Diazepam has been reported to inhibit both the tremor and mechanism of cerebellar cyclic GMP caused by harmaline by a neurotransmission in the cerebellum. Because of the similarities between ethanol and benzodiazepines, the effects of ethanol on harmaline-induced tremor and increase of cerebellar cyclic GMP were studied. Ethanol inhibited harmaline-induced tremor at doses as low as 0.1 g/kg. At this low dose, however, a dissociation between inhibition of harmaline tremor and inhibition of the harmaline-induced increase of cerebellar cyclic GMP was observed.

Cerebral blood flow after treatment with ORG-2766, a potent analog of ACTH 4--9.

Regional cerebral blood flows (rCBF) were measured in conscious, male rats at 10, 30, 60 min and 24 hr after intravenous administration of a potent, behaviorally active analog of ACTH/MSH 4--9 (ORG-2766). Flows in the basal ganglia, hippocampus, septal area and frontal cortex were depressed significantly throughout the 60 min postinjection period. HYpothalamic and parietal flows were depressed at 10 and 30 min, but recovered by 60 min, whereas flow to the cerebellum was depressed between 30 and 60 min postinjection. The least changed and therefore relatively better perfused area throughout the first 60 min period was the occipital cortex. By contrast, at 24 hr, when perfusion of all brain regions had returned to near control levels, flow to the occipital cortex was elevated. During the first hour after treatment with either ORG-2766 or alphaMSH the patterns of regional circulation in the brain were qualitatively the same. The data suggest that ORG-2766 and, probably, alpha MSH trigger serially linked neurophysiologic changes in the brain lasting at least 24 hr, which organize the behavioral actions of this class of peptides on memory and attentional processes.

Effect of diesel particulate exposure on adenylate and guanylate cyclase of rat and guinea pig liver and lung.

An examination of the effects of 250 or 1500 micrograms m-3 concentrations of diesel particulate from diesel exhaust on the activity of adenylate or guanylate cyclase was undertaken using liver and lung tissue of rats and guinea pigs. These membrane and cytosolic enzymes were selected to screen for functional or regulatory alterations in these tissues. The studies for adenylate cyclase used the microsomal membrane fraction of each tissue; for guanylate cyclase, the microsomal membrane and supernate fractions were used. Basal and fluoride-stimulated adenylate cyclase activity were measured. Basal and sodium azide-stimulated guanylate cyclase activity were also determined. The basal activity of rat liver adenylate cyclase is generally unchanged throughout 52 weeks of diesel exposure. Stimulated adenylate cyclase shows an age-related decrease for all animal treatments throughout the study. Changes in enzyme activity occurred at 12 weeks and 52 weeks after 1500 micrograms m-3 exposure. Soluble stimulated guinea pig lung guanylate cyclase was first increased (6 weeks) and then decreased (24 weeks) by diesel exposure. At 52 weeks, there was no change. The data suggest the following trends: (1) an increased basal adenylate cyclase in the rat lung; (2) an age-related decrease in adenylate cyclase activity in rat liver, and (3) a biphasic exposure-related response of soluble guanylate cyclase for the guinea pig lung during the first 24 weeks, but no change at 52 weeks. In general, however, these studies suggest that diesel exposure does not substantially alter either of these intracellular regulating enzymes.

Performance characteristics of a multimedia system for clinical data management.

This study describes a multimedia, computer-based system developed to record and retrieve clinical images, associated dictated commentaries, and textual and numeric data from patients with cancer or hematologic diseases. Objective performance was analyzed by (1) success rate for data input and retrieval, (2) speed of image retrieval, and (3) quality of stored images. During a 1-year period, 587 diagnostic images were recorded with dictated or typed summaries of pertinent clinical information. Subsequent retrieval of stored data was performed three times (n = 1761) with 100% reliability. The average time for retrieval of random images was 6.4 seconds (SD = 1.7 seconds), including operator time. Image quality was scored by a four-point scale: the average score was 3.58 (SD = 0.5), and all images displayed sufficient quality to represent a wide range of diagnoses. These data demonstrate that multimedia computer technologies can enhance the organization and management of clinical images needed for medical education, clinical trials, and daily care of patients with cancer and hematologic diseases.

Effect of pyridines on phenotypic properties of Bordetella pertussis.

Several conditions of growth of Bordetella pertussis cause a reversible phenotypic alteration in properties termed modulation. Growth in medium containing nicotinic acid induces normal (X-mode) cells to change to modulated (C-mode) cells. We examined several pyridines and compounds resembling pyridines for their ability to affect modulation, using envelope protein patterns and serological reactivity as indicators of modulation. We found that 6-chloronicotinic acid and quinaldic acid were more effective modulating stimuli than was nicotinic acid on a molar basis. Both 2-chloronicotinamide and isoniazid interfered with nicotinic acid-induced modulation, and can be called antimodulators. Picolinic acid inhibited growth.

Purification and characterization of the mucinase of Vibrio cholerae.

Mucinase from Vibrio cholerae strain CA401 was purified by precipitation with ammonium sulfate and chromatography on a column of BioGel P-100 (Bio-Rad Laboratories, Richmond, California). Ovomucinase, intestinal mucinase, and protease appeared as two peaks of slightly different molecular weights, comigrated in nondenaturing polyacrylamide gel electrophoresis, demonstrated similar patterns of inhibition by heavy metals, and were inhibited by antiserum to purified mucinase. Both molecular weight forms exhibited similar properties, which were identical to those of fresh culture supernatants. Specific activity was only slightly increased by purification. Antiserum to mucinase passively protected infant mice from diarrhea due to V. cholerae.

Isolation and characterization of protease-deficient mutants of vibrio cholerae.

Mutants of Vibrio cholerae that were deficient in protease production were isolated by picking clones form gelatin or casein plates which showed reduced zones of proteolysis. All mutants showed reduced ability to degrade complex proteins (casein and gelatin), and those tested were deficient in ability to degrade chicken egg ovomucin. Some of the mutants demonstrated a decrease in neuraminidase activity. Almost all mutants showed a dramatic loss of virulence in the infant mouse, although toxin was still produced. A partial revertant, to protease-proficient, demonstrated a simultaneous increase in neuraminidase activity and also an increase in mouse virulence. The strains described had a variety of phenotypes.

A model for enzymatic isomerization of D-glucose to D-fructose in a batch reactor.

Using whole cells containing glucose isomerase, mathematical models for the enzymatic conversion of D-glucose to D-fructose and for the inactivation of the enzyme catalyst have been postulated and verified experimentally. The heat of reaction, the equilibrium constant, and the individual rate constants and their activation energies have been estimated. The model can be used to predict the time course for the enzymatic production of fructose in a batch reactor within the tested experimental range of 40-80 degrees C.

Characterization of Vibrio cholerae protease activities with peptide digest analysis.

A simple method for the analysis of microbial proteases is described that was used to characterize the proteolytic activities of various Vibrio cholerae isolates. This method utilized the unique peptides generated from the degradation of a standard protein by proteases of various specificities. These peptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The unique patterns of peptides seen in gels can be used to type proteases according to their relative specificities. Culture supernatants of V. cholerae isolates from a variety of environmental and human sources were analyzed for the presence of a protease previously isolated and characterized in this laboratory from V. cholerae strain CA401. Supernatants from most isolates showing dimethyl casein proteolytic activity exhibited the presence of enzymes similar to the CA401 protease in their peptide digest patterns against bovine serum albumin and in their immunological reactivities. The probable widespread presence of this virulence-associated protease in V. cholerae isolates is discussed.

An automated microprocessor-controlled data collection device for use with the Technicon AutoAnalyzer system.

An automated microprocessor-controlled data collection system for use with a Technicon AutoAnalyzer in a research or clinical laboratory is described. The collection system can digitize an analog output from the AutoAnalyzer, perform mathematical operations on this digital data, detect and record peak-trough data, and perform these data acquisition and mathematical data processing operations in parallel. The data collection system can be interfaced to a variety of host computers for further data analysis. The accuracy of the data collection system was tested and compared to existing procedures using an analysis of bovine serum albumin protein. The correlation coefficient for the comparison of the results was 0.99998. Hence, the automated system is as accurate as previous methods, but is considerably faster, more efficient, and, in comparison to existing devices, less expensive.

Alpha 1-antitrypsin and lysozyme in fibrous papules and angiofibromas.

The large stellate and polygonal cells observed in eleven fibrous papules and two angiofibromas were examined immunohistochemically for alpha 1-antitrypsin and lysozyme. The positive findings suggest that these cells are related to histiocytes rather than nevomelanocytes.