Quantification methodology for peripheral quantitative computed tomography (PQCT) data using public domain software.

OBJECTIVE: To develop a method for viewing, processing and acquiring 3-dimensional volumetric data from existing ovine spinal peripheral quantitative computed tomography (PQCT) scans of posterior lateral fusion. DESIGN: An image processing development study. BACKGROUND: Existing medical image viewing software can be expensive and difficult to adapt to meet specific research needs. The goals of this study were to produce volume rendering of PQCT scans through processing, masking, and segmentation using public domain software with established source code. METHODS: Raw data files (DICOM format) of 32 PQCT scans from animals receiving spine fusion were obtained. Metal hardware was removed from the images by masks and image segmentation. Calculation of bone macro-architecture volumetric data was performed on right and left sides of spines to quantify fusion volume in normalized segments between transverse processes. RESULTS: Images were acquired and opened. Application of image processing techniques made it possible to remove surgical hardware from original images with minimal loss of original PQCT data. Volumes were calculated and normalized to gray-scale of total bone throughout individual selected segments. CONCLUSION: Using public domain software is a cost effective means to view, process, and manipulate PQCT data. Bone macro-architecture can provide quantitative volumetric contributions to ascertain the role of structure on mechanical function.

The Brucella abortus Lon functions as a generalized stress response protease and is required for wild-type virulence in BALB/c mice.

The gene encoding a Lon protease homologue has been cloned from Brucella abortus. The putative Brucella abortus Lon shares > 60% amino acid identity with its Escherichia coli counterpart and the recombinant form of this protein restores the capacity of an Escherichia coli lon mutant to resist killing by ultraviolet irradiation and regulate the expression of a cpsB:lacZ fusion to wild-type levels. A sigma32 type promoter was identified upstream of the predicted lon coding region and Northern analysis revealed that transcription of the native Brucella abortus lon increases in response to heat shock and other environmental stresses. ATP-dependent proteolytic activity was also demonstrated for purified recombinant Lon. To evaluate the capacity of the Brucella abortus Lon homologue to function as a stress response protease, the majority of the lon coding region was removed from virulent strain Brucella abortus 2308 via allelic exchange. In contrast to the parent strain, the Brucella abortus lon mutant, designated GR106, was impaired in its capacity to form isolated colonies on solid medium at 41 degrees C and displayed an increased sensitivity to killing by puromycin and H2O2. GR106 also displayed reduced survival in cultured murine macrophages and significant attenuation in BALB/c mice at 1 week post infection compared with the virulent parental strain. Beginning at 2 weeks and continuing for 6 weeks post infection, however, GR106 and 2308 displayed equivalent spleen and liver colonization levels in mice. These findings suggest that the Brucella abortus Lon homologue functions as a stress response protease that is required for wild-type virulence during the initial stages of infection in the mouse model, but is not essential for the establishment and maintenance of chronic infection in this host.

Identification and characterization of a 14-kilodalton Brucella abortus protein reactive with antibodies from naturally and experimentally infected hosts and T lymphocytes from experimentally infected BALB/c mice.

A low-molecular-weight recombinant Brucella abortus protein reactive with antibodies from a variety of naturally and experimentally infected hosts and T lymphocytes from experimentally infected mice was identified and given the designation BA14K. The gene encoding BA14K was cloned and characterized, and the predicted amino acid sequence of this immunoreactive protein showed no significant homology with previously described proteins. Sequences homologous to the cloned fragment encoding BA14K were identified by Southern blot analysis of genomic DNAs from representatives of all of the currently recognized Brucella species. Studies employing BA14K should contribute to our efforts to better understand the antigenic specificity of protective immunity to brucellosis.

Cloning and nucleotide sequence analysis of a Brucella abortus gene encoding an 18 kDa immunoreactive protein.

A DNA fragment encoding an approximately 18 kDa protein from Brucella abortus strain 2308 was cloned and expressed in Escherichia coli. This recombinant protein, designated BA18K, reacted in Western blot analysis with sera obtained from experimentally and naturally infected animals including mice, goats, dogs and humans. Restriction enzyme analysis of the plasmid (pBA28) encoding BA18K revealed the presence of an approximately 8.7 kbp Sau3A genomic DNA fragment within the vector and subsequent subcloning and Western blot analysis limited the region encoding BA18K to an approximately 3.0 kbp Pst 1 DNA fragment. DNA sequence analysis of this region identified an open reading frame capable of encoding a protein of 177 amino acids with a predicted relative molecular mass of 17529. Comparison of the deduced amino acid sequence of BA18K with those in the protein sequence databases yielded no homology with previously described proteins from other bacterial genera. These searches did, however, indicate that BA18K is identical to the previously described outer membrane protein (OMP) from B. abortus strain 544 designated Omp 19.

A putative integrase gene defines the distal end of a large cluster of ToxR-regulated colonization genes in Vibrio cholerae.

A large cluster of virulence genes encoding proteins involved in Vibrio cholerae accessory colonization factor (ACF) expression and toxin-coregulated pilus (TCP) biogenesis is flanked by sequences that resemble bacteriophage attachment (att) half-sites. Adjacent to the attL-like site is a gene (int) that encodes a protein related to the integrase family of site-specific recombinases. The putative vibrio integrase appears to be most closely related to the Escherichia coli cryptic prophage (CP4-57) integrase protein (52% identity, 73% similarity). Genomic analysis of numerous V. cholerae strains (O1, non-O1 and O139) revealed that only vibrios capable of causing epidemic Asiatic cholera possess the TCP-ACF colonization gene cluster in association with the integrase. The fact that the integrase gene is absent in avirulent strains suggests that epidemic strains of V. cholerae obtained the TCP-ACF colonization gene cluster via horizontal transfer.

Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.

Four new antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector pBBR1MCS have been constructed. These new plasmids have several advantages over many of the currently available bhr vectors in that: (i) they are relatively small (< 5.3 kb), (ii) they possess an extended multiple cloning site (MCS), (iii) they allow direct selection of recombinant plasmid molecules in Escherichia coli via disruption of the LacZ alpha peptide, (iv) they are mobilizable when the RK2 transfer functions are provided in trans and (v) they are compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons.

Isolation and characterization of the Vibrio cholerae acfA gene, required for efficient intestinal colonization.

The nucleotide sequence of the Vibrio cholerae acfA gene (encoding an accessory colonization factor) has been determined. Sequence analysis revealed the presence of an open reading frame of 215 amino acids with a characteristic signal peptidase I (SPI) cleavage site at the N terminus. Electrophoretic analysis of proteins synthesized by Escherichia coli cells, following T7 promoter/RNA polymerase-directed expression of acfA, revealed a 23-kDa protein corresponding to the mature form of AcfA. The T7 expression system also showed that, in the presence of known SPI inhibitors, a 25-kDa unprocessed form of AcfA is produced.

In vivo and in vitro stability of the broad-host-range cloning vector pBBR1MCS in six Brucella species.

Previous studies have shown that the broad-host-range plasmid pBBR1MCS can be used for genetic complementation in Brucella abortus. To extend these observations, the in vivo and in vitro stability of pBBR1MCS was evaluated in the six currently recognized species of the genus Brucella. pBBR1MCS was readily introduced into all of the strains tested by electroporation and was stably maintained in broth cultures without antibiotic selection during five serial passages over a 10-day period. Furthermore, isolates of all six Brucella strains containing pBBR1MCS obtained from the spleens of BALB/c mice 1 week postinfection maintained the plasmid. Although pBBR1MCS maintains the mobilization locus present in the parental plasmid pBBR1CM, attempts to detect transfer of pBBR1MCS between Brucella strains by conjugation were unsuccessful. These results demonstrate the in vitro and in vivo stability of pBBR1MCS in Brucella spp. and reinforce the usefulness of this cloning vector for the genetic analysis of these organisms.

Identification of a ToxR-activated gene, tagE, that lies within the accessory colonization factor gene cluster of Vibrio cholerae O395.

The nucleotide (nt) sequence has been determined for a Vibrio cholerae ToxR-activated gene designated tagE that is located within a cluster of genes required for efficient intestinal colonization. The tagE gene encompasses 909 nt and is predicted to encode a 303-amino-acid (aa) protein with an estimated molecular mass of 34,468 Da. Computer-assisted similarity searches revealed that TagE possesses aa sequence similarity with Escherichia coli OrfU and Staphylococcus simulans lysostaphin, two proteins that are involved in cell-wall biosynthesis and peptidoglycan degradation, respectively. The role, if any, that TagE plays in the accessory colonization factor phenotype is currently under investigation.

Characterization and genetic complementation of a Brucella abortus high-temperature-requirement A (htrA) deletion mutant.

In order to evaluate the biological function of the Brucella abortus high-temperature-requirement A (HtrA) stress response protein homolog, the majority of the htrA gene was deleted from the chromosome of B. abortus 2308 via gene replacement. In contrast to the parental strain, the resulting htrA deletion mutant, designated PHE1, failed to grow on solid medium at 40 degrees C and demonstrated increased sensitivity to killing by H2O2 and O2- in disk sensitivity assays. BALB/c mice were infected with strains 2308 and PHE1 to assess the effect of the htrA mutation on virulence, and significantly fewer brucellae were recovered from the spleens of mice infected with PHE1 than from those of mice infected with 2308 at 1 week postinfection. Genetic complementation studies were performed to confirm the relationship between the htrA mutation and the phenotype observed for PHE1. Plasmid pRIE1 was constructed by inserting a 1.9-kb EcoRI fragment encoding the B. abortus htrA gene into the broad-host-range plasmid pBBR1MCS. Introduction of pRIE1 into PHE1 relieved the temperature- and H2O2-sensitive phenotypes of this mutant in vitro, and PHE1(pRIE1) colonized the spleens of BALB/c mice at levels equivalent to those of the parental 2308 strain at 1 week postinfection. These results support our previous proposal that the B. abortus htrA gene product functions as a stress response protein and further suggest that this protein contributes to virulence. These studies also demonstrate the utility of the broad-host-range plasmid pBBR1MCS for genetic complementation studies in Brucella spp., establishing a key reagent for more detailed genetic analysis of this important zoonotic pathogen.

Sequence analysis of the Vibrio cholerae acfD gene reveals the presence of an overlapping reading frame, orfZ, which encodes a protein that shares sequence similarity to the FliA and FliC products of Salmonella.

The nucleotide (nt) sequence of the Vibrio cholerae acfD gene (encoding an accessory colonization factor) has been determined. The acfD gene encompasses 254 nt that are predicted to encode an 88-amino-acid (aa) protein. Additionally, an open reading frame of 184 aa, designated orfZ, was detected that overlaps the 3' end of acfD by 45 nt. Computer-assisted homology searches revealed that OrfZ possesses aa sequence similarity to the C terminus of the fliC product of Salmonella muenchen and S. rubislaw. Additionally, OrfZ shares limited aa sequence similarity to a region of the S. typhimurium fliA product. Interestingly, a V. cholerae acfD::TnphoA mutant demonstrates reduced motility and an altered swarming phenotype in semi-solid media when compared to wild-type V. cholerae. The possibility that the reduced motility and altered morphology result from polar effects on orfZ expression is currently being investigated.

The Vibrio cholerae acfB colonization determinant encodes an inner membrane protein that is related to a family of signal-transducing proteins.

Vibrio cholerae accessory colonization factor genes (acfA, B, C, and D) are required for efficient intestinal colonization. Expression of acf genes is under the control of a regulatory cascade that also directs the synthesis of cholera toxin and proteins involved in the biogenesis of the toxin-coregulated pilus. The gene for acfB was cloned by using an acfB::TnphoA fusion junction to probe a V. cholerae O395 bacteriophage lambda library. DNA sequence analysis revealed that acfB is predicted to encode a 626-amino-acid protein related to the V. cholerae HlyB and TcpI proteins. These three Vibrio proteins have amino acid sequence similarity in a region highly conserved among bacterial methyl-accepting chemotaxis proteins. Analysis of the predicted AcfB amino acid sequence suggests that this colonization determinant possesses a membrane topology and domain organization similar to those of methyl-accepting chemotaxis proteins. Heterologous expression of acfB in Escherichia coli generates four polypeptide species with apparent molecular masses of 34, 35, 74, and 75 kDa. The 74- and 75-kDa proteins appear to represent modified forms of the full-length AcfB protein. The 34- and 35-kDa polypeptide species most likely correspond to a C-terminal 274-amino-acid polypeptide that results from internal translation initiation of acfB mRNA. Localization studies with AcfB-PhoA hybrid proteins indicate that AcfB resides in the V. cholerae inner membrane. V. cholerae acfB::TnphoA mutants display an altered motility phenotype in semisolid agar. The relationship between AcfB and Vibrio motility and the amino acid similarities between AcfB and chemotaxis signal-transducing proteins suggest that AcfB may interact with the V. cholerae chemotaxis machinery. The data presented in this report provide preliminary evidence that acfB encodes an environmental sensor/signal-transducing protein involved in V. cholerae colonization.