Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene

B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.     

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.     

Structure-function analysis of recombinant substrate protein 22 kDa (SP-22). A mitochondrial 2-CYS peroxiredoxin organized as a decameric toroid

Bovine mitochondrial SP-22 is a member of the peroxiredoxin family of peroxidases. It belongs to the peroxiredoxin 2-Cys subgroup containing three cysteines at positions 47, 66, and 168. The cloning and overexpression in Escherichia coli of recombinant wild type SP-22 and its three cysteine mutants (C47S, C66S, and C168S) are reported. Purified His-tagged SP-22 was fully active with Cys-47 being confirmed as the catalytic residue. The enzyme forms a stable decameric toroid consisting of five basic dimeric units containing intermolecular disulfide bonds linking the catalytically active Cys-47 of one subunit and Cys-168 of the adjacent monomer. The disulfide bonds are not required for overall structural integrity. The toroidal units have average external and internal diameters of 15 and 7 nm, respectively, and can form stacks in a lateral arrangement of two or three rings. C47S had a pronounced tendency to stack in long tubular structures containing up to 60 rings. Further unusual structural features are the presence of radial spikes projecting from the external surface and ordered electron-dense material within the central cavity of the toroid.     

Truncation of the carboxyl terminus of the dihydropyridine receptor beta1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes

We investigated the contribution of the carboxyl terminus region of the beta1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling. cDNA-transfected beta1 KO myotubes were voltage clamped, and Ca(2+) transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of beta2a and a carboxyl terminus half of beta1a (beta2a 1-287/beta1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the beta1a-specific carboxyl terminus (Delta7, Delta21, Delta29, Delta35, and Delta60). Ca(2+) transients recovered by the control chimera have a sigmoidal Ca(2+) fluorescence (DeltaF/F) versus voltage curve with saturation at potentials more positive than +30 mV, independent of external Ca(2+) and stimulus duration. In contrast, the amplitude of Ca(2+) transients expressed by the truncated variants varied with the duration of the pulse, and for Delta29, Delta35, and Delta60, also varied with external Ca(2+) concentration. For Delta7 and Delta21, a 50-ms depolarization produced a sigmoidal DeltaF/F versus voltage curve with a lower than control maximum fluorescence. Moreover, for Delta29, Delta35, and Delta60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the DeltaF/F versus voltage curve, from sigmoidal to bell-shaped, with a maximum at approximately +30 mV. The change in voltage dependence, together with the external Ca(2+) dependence and additional controls with ryanodine, indicated a loss of skeletal-type EC coupling and the emergence of an EC coupling component triggered by the Ca(2+) current. Analyses of d(DeltaF/F)/dt showed that the rate of cytosolic Ca(2+) increase during the Ca(2+) transient was fivefold faster for the control chimera than for the severely truncated variants (Delta29, Delta35, and Delta60) and was consistent with the kinetics of the DHPR Ca(2+) current. In summary, absence of the beta1a-specific carboxyl terminus (last 29 to 60 residues of the control chimera) results in a loss of the fast component of the Ca(2+) transient, bending of the DeltaF/F versus voltage curve, and emergence of EC coupling triggered by the Ca(2+) current. The studies underscore the essential role of the carboxyl terminus region of the DHPR beta1a subunit in fast voltage dependent EC coupling in skeletal myotubes.     

Structural characterisation of a perdeuteriomethylated exopolysaccharide by NMR spectroscopy: characterisation of the novel exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus EU23

The exopolysaccharide (EPS) from Lactobacillus delbrueckii subsp. bulgaricus EU23 was perdeuteriomethylated and the perdeuteriomethylated EPS (pdm-EPS) purified by elution from a C(18) Sep-Pak cartridge. Both 1D and 2D NMR spectra were recorded for the pdm-EPS and these were interpreted to provide assignments for the individual 1H and 13C resonances of the sugar residues of the repeating unit. Using a combination of the results from monomer analysis and linkage analysis of the native EPS and the ROESY and HMBC NMR spectra of the pdm-EPS the following structure has been determined for the repeating unit:A process for characterising polysaccharides having low solubility in aqueous solution is reported.     

Characterization of extradiol dioxygenases from a polychlorinated biphenyl-degrading strain that possess higher specificities for chlorinated metabolites

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBDLB400; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2',6'-dichloro-2,3-dihydroxybiphenyl (2',6'-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBDP6-I and DHBDP6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBDLB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBDP6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3'-Cl DHB approximately DHB approximately 4'-Cl DHB > 2'-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBDP6-I were among the best for DHBDP6-III (4-Cl DHB > 5-Cl DHB approximately 6-Cl DHB approximately 3'-Cl DHB > DHB > 2'-Cl DHB approximately 4'-Cl DHB; DHBDP6-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBDP6-I and 2'-Cl DHB for DHBDP6-III. As observed in DHBDLB400, chloro substituents influenced the reactivity of the dioxygenases with O2. For example, the apparent specificities of DHBDP6-I and DHBDP6-III for O2 in the presence of 2'-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBDP6-I and DHBDP6-III shared the relative inability of DHBDLB400 to cleave 2',6'-diCl DHB (apparent catalytic constants of 0.088 +/- 0.004 and 0.069 +/- 0.002 s(-1), respectively). However, these isozymes had remarkably different apparent K(m) values for this compound (0.007 +/- 0.001, 0.14 +/- 0.01, and 3.9 +/- 0.4 micro M for DHBDLB400, DHBDP6-I, and DHBDP6-III, respectively). The markedly different reactivities of DHBDP6-I and DHBDP6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.     

Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm

The biofilm proteome of a dairy-associated Bacillus cereus strain (B. cereus 5) was investigated. Biofilm biomass of sufficient concentration for 2D-PAGE was obtained by growing the culture in the presence of glass wool. B. cereus 5 readily attached to the glass wool and biofilms formed within 18 h. The biofilm proteome of whole-cell proteins revealed that 10 proteins were synthesized as a result of surface attachment of which four were unique to the biofilm profile. Seven proteins appeared to be absent in the biofilm profile. The altered proteomes indicated that changes took place in the regulation of protein expression when B. cereus 5 cells attached to surfaces.