Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida

The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0%. The protein crystallizes in the space group I222 with one subunit in the asymmetric unit. The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A. The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000. The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues. The structure was determined without a bound substrate or substrate analogue. Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423. On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily [Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R. J., Barrett, W. C., Reed, G. J., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) Biochemistry 35, 16489-16501]. This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase. Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR. The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed.     

The repertoire of Anaplasma marginale antigens recognized by CD4(+) T-lymphocyte clones from protectively immunized cattle is diverse and includes major surface protein 2 (MSP-2) and MSP-3

Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain of A. marginale resulted in protective immunity that correlated with a memory CD4(+) T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406-5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and other A. marginale antigens and to determine conservation of Th cell epitopes among genetically distinct A. marginale strains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida strain. Several clones responded to MSP-2, MSP-3, or both. All of these MSP-2- or MSP-3-specific clones and the majority of other clones that did not respond to MSPs recognized all bovine blood-passaged strains of A. marginale. These results demonstrate conservation of certain Th cell epitopes between MSP-2 and MSP-3 and show that Th cell epitopes in MSP-2, MSP-3, and undefined antigens are conserved among strains of A. marginale. Of seven clones that responded to the blood-passaged Virginia strain, two did not recognize antigen prepared from this strain cultured in tick cells, suggesting differences in the antigenic composition between these stages. Analysis of the cytokines expressed by the Th cells revealed that all clones expressed gamma interferon and tumor necrosis factor alpha, and most coexpressed interleukin-4. Our results provide a rationale for identifying Th cell epitopes conserved among different strains of A. marginale for inclusion in a nucleic acid or recombinant protein vaccine.     

A selective role of brainstem noradrenergic neurons in oxytocin release from the neurohypophysis following noxious stimuli in the rat

Infantile nephropathic cystinosis is a genetic metabolic disorder in which the amino acid cystine accumulates in various organs, including the kidney, cornea, thyroid, and brain. Despite normal intellect, individuals with cystinosis may have specific impairments in the processing of visual information. To examine further the specific types of deficits in visual processing found in individuals with cystinosis, we administered the Development Test of Visual-motor Integration to 26 children with cystinosis (4 to 16 yr. old) and 26 matched controls. The cystinosis group achieved a significantly lower standard score, raw score, and mean ceiling than did the control group. Qualitative analyses showed that in the cystinosis group, size within errors and rotation errors were more prevalent than in the control group. Correlational analyses showed that with advancing age, the cystinosis subjects tended to fall further behind their chronological age. Our data, together with the findings of previous studies, suggest that the visuospatial difficulties in children with cystinosis may be due to inadequate perception or processing of visually presented information. Furthermore, the increasing discrepancy with age may reflect a progressive cognitive impairment, possibly as a result of cystine accumulation in the brain over time.     

Overlapping functions of components of a bacterial Sec-independent protein export pathway

We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.     

The effect of benomyl on neurite outgrowth in mouse NB2A and human SH-SY5Y neuroblastoma cells in vitro

The commercial fungicide methyl 1-[(butylamino) carbonyl]-1H-benzimidazol-2-ylcarbamate (benomyl) is teratogenic in rats. Its mode of action is believed to be related to its ability to inhibit the polymerization of brain tubulin. In this study its effects were studied in cultured neuronal cells during differentiation and neurite outgrowth. Mouse NB2a and human SH-SY5Y neuroblastoma cells were induced to differentiate by addition of dibutyryl cyclic AMP and at the same time were exposed to various concentrations of benomyl. Benomyl significantly inhibited neurite outgrowth in both cell lines at concentrations of 10(-8) M and above with IC50 values of 5.9 x 10(-7) M and 1.0 x 10(-6) M in the NB2a and SH-SY5Y cells respectively. The results show that benomyl inhibits neuronal cell differentiation at concentrations likely to be achieved during the development of fetal abnormalities in rats in vivo.     

Anti-oxidant enzymes, gamma-glutamyl transpeptidase and lipid peroxidation in kidney of rats exposed to cigarette smoke

Glutathione reductase (GR), glutathione peroxidase (GSH Px) and catalase (Cat) were determined in the kidneys of rats exposed to cigarette smoke for 3 months. Activity of the brush-border enzyme gamma-glutamyl transpeptidase (GGT), glutathione (GSH) and lipid peroxide levels (LPX) were also estimated in both, kidney homogenates and urine. Activities of GR, Cat, GGT and the levels of GSH were decreased in the kidney. However, the activities of GSH Px and LPX levels were increased. Urinary excretion of GGT, GSH and LPX were also higher. Fall in the activity of GR and rise in the activity of GSH Px, may perturb the reduced glutathione/oxidised glutathione ratio, which in turn could lead to increased LPX seen in chronic cigarette smoke exposure.     

General education teachers' knowledge and self-reported use of classroom interventions for working with difficult-to-teach students: Implications for consultation, prereferral intervention and inclusive services.

Twenty 3rd- and 4th-grade teachers were interviewed about working with difficult-to-teach students (e.g, children with specific learning disabilities, mild mental handicaps, behavior disorders) in their classrooms. Using a referral case and a standardized vignette, teachers described problems, goals, interventions, data collection, and consultation practices employed across prereferral, prereferral Intervention, referral, and post-referral phases of service delivery. Most participants demonstrated only vague knowledge of classroom interventions. Few accommodations were made in general classrooms for mildly-handicapped children, particularly following the prereferral phase. Participants consulted primarily with other general educators and secondarily with special educators. Consultation with school psychologists was unusual. Discussion focuses on implications for consultation, prereferral intervention, and inclusive services. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Myxoma of the left atrial appendage mimicking thrombus during aortic valve replacement

A previously well, 48-year-old female presented with a short history of respiratory distress and fever for which no cause could be found. Open lung biopsy was not contributory. She died within nine days of presentation of hypoxia and multi-organ failure. Post mortem examination revealed the pattern of desquamative interstitial pneumonitis.     

Exophthalmos and facial swelling: a case of limited Churg-Strauss syndrome

We report a case of vasculitis causing facial swelling and exophthalmos which on clinical and histological grounds is thought to be limited Churg-Strauss syndrome. An excellent response was achieved to high doses of systemic steroids and cyclophosphamide.