Computed tomographic arthrography is a useful adjunct to survey computed tomography and arthroscopic evaluation of the canine shoulder joint.

The aim of this retrospective, methods comparison study was to assess the diagnostic utility of computed tomographic arthrography in the assessment of various intraarticular shoulder pathologies in dogs in comparison with survey computed tomography (CT), using arthroscopic examination as the reference standard. Computed tomography, computed tomographic arthrography, and arthroscopic findings of 46 scapulohumeral joints of dogs with forelimb lameness were reviewed retrospectively. Predefined sites were assessed for the presence or absence of disease. If a lesion was present, a prespecified pathology was designated. Computed tomographic arthrography was found to be a safe technique which provided a superior diagnostic efficacy relative to survey CT for the assessment of the biceps tendon and biceps tendon sheath (sensitivity 71%, specificity 75%, positive likelihood ratio 2.9, negative likelihood ratio 0.38) and humeral head cartilage (sensitivity 65%, specificity 97%, positive likelihood ratio 19, negative likelihood ratio 0.37). Computed tomography and computed tomographic arthrography provided additional diagnostic information to arthroscopy in regard to osteophytosis, subchondral defects, and joint mice. Computed tomographic arthrography alone was of limited diagnostic value for assessment of the medial and lateral glenohumeral ligaments (sensitivity 13% and 0%, specificity 1% and 78%, positive likelihood ratios unmeasurable and 0, negative likelihood ratios 0.88 and 1.29, respectively) and the subscapularis tendon (sensitivity 14%, specificity 98%, positive likelihood ratio 5.7, negative likelihood ratio 0.88). Computed tomographic arthrography is therefore a useful adjunct to survey CT and arthroscopic evaluation of the canine shoulder joint, however, is not a replacement for these techniques.

Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains.

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget's disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

Correction to: Increased heterologous production of the antitumoral polyketide mithramycin a by engineered Streptomyces lividans TK24 strains.

The original publication contains error error in the Materials and Methods section and in the acknowledgement section.

Lipase-catalyzed preparation of chromomycin A3 analogues and biological evaluation for anticancer activity.

Several acyl derivatives of the aureolic acid chromomycin A(3) were obtained via lipase-catalyzed acylation. Lipase B from Candida antarctica (CAL-B) was found to be the only active biocatalyst, directing the acylation regioselectively towards the terminal secondary hydroxyl group of the aglycone side chain. All new chromomycin A(3) derivatives showed antitumor activity at the micromolar or lower level concentration. Particularly, chromomycin A(3) 4'-vinyladipate showed 3-5 times higher activity against the four tumor cell lines assayed as compared to chromomycin A(3).

A novel mithramycin analogue with high antitumor activity and less toxicity generated by combinatorial biosynthesis.

Mithramycin is an antitumor compound produced by Streptomyces argillaceus that has been used for the treatment of several types of tumors and hypercalcaemia processes. However, its use in humans has been limited because of its side effects. Using combinatorial biosynthesis approaches, we have generated seven new mithramycin derivatives, which differ from the parental compound in the sugar profile or in both the sugar profile and the 3-side chain. From these studies three novel derivatives were identified, demycarosyl-3D-ß-d-digitoxosylmithramycin SK, demycarosylmithramycin SDK, and demycarosyl-3D-ß-d-digitoxosylmithramycin SDK, which show high antitumor activity. The first one, which combines two structural features previously found to improve pharmacological behavior, was generated following two different strategies, and it showed less toxicity than mithramycin. Preliminary in vivo evaluation of its antitumor activity through hollow fiber assays, and in subcutaneous colon and melanoma cancers xenografts models, suggests that demycarosyl-3D-ß-d-digitoxosylmithramycin SK could be a promising antitumor agent worthy of further investigation.

Intraobserver, interobserver, and intermethod agreement for results of myelography, computed tomography-myelography, and low-field magnetic resonance imaging in dogs with disk-associated wobbler syndrome.

OBJECTIVE: To determine intraobserver, interobserver, and intermethod agreement for results of myelography, computed tomography-myelography (CTM), and low-field magnetic resonance imaging (MRI) in dogs with disk-associated wobbler syndrome (DAWS). DESIGN: Prospective cross-sectional study. ANIMALS: 22 dogs with DAWS. PROCEDURES: All dogs underwent myelography, CTM, and low-field MRI. Each imaging study was interpreted twice by 4 observers who were blinded to signalment and clinical information of the patients. The following variables were assessed by all 3 techniques: number, site, and direction of spinal cord compressions; narrowed intervertebral disk spaces; vertebral body abnormalities; spondylosis deformans; and abnormal articular facets. Intervertebral foraminal stenosis was assessed on CTM and MRI images. Intraobserver, interobserver, and intermethod agreement were calculated by κ and weighted κ statistics. RESULTS: There was very good to good intraobserver agreement for most variables assessed by myelography and only moderate intraobserver agreement for most variables assessed by CTM and low-field MRI. There was moderate to fair interobserver and intermethod agreement for most variables assessed by the 3 diagnostic techniques. There was very good or good intraobserver, interobserver, or intermethod agreement for the site and direction of the worst spinal cord compression as assessed by all the imaging modalities; abnormal articular facets and intervertebral foraminal stenosis were the least reliably assessed variables, with poor interobserver agreement regardless of imaging modality used. CONCLUSIONS AND CLINICAL RELEVANCE: There was considerable variation in image interpretation among observers and between use of various imaging modalities; these imaging techniques should be considered complementary in assessment of dogs with DAWS.

The chromomycin CmmA acetyltransferase: a membrane-bound enzyme as a tool for increasing structural diversity of the antitumour mithramycin.

Mithramycin and chromomycin A(3) are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl-CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.

Intraobserver and interobserver agreement for results of low-field magnetic resonance imaging in dogs with and without clinical signs of disk-associated wobbler syndrome.

OBJECTIVE: To determine interobserver and intraobserver agreement for results of low-field magnetic resonance imaging (MRI) in dogs with and without disk-associated wobbler syndrome (DAWS). DESIGN: Validation study. ANIMALS: 21 dogs with and 23 dogs without clinical signs of DAWS. PROCEDURES: For each dog, MRI of the cervical vertebral column was performed. The MRI studies were presented in a randomized sequence to 4 board-certified radiologists blinded to clinical status. Observers assessed degree of disk degeneration, disk-associated and dorsal compression, alterations in intraspinal signal intensity (ISI), vertebral body abnormalities, and new bone formation and categorized each study as originating from a clinically affected or clinically normal dog. Interobserver agreement was calculated for 44 initial measurements for each observer. Intraobserver agreement was calculated for 11 replicate measurements for each observer. RESULTS: There was good interobserver agreement for ratings of disk degeneration and vertebral body abnormalities and moderate interobserver agreement for ratings of disk-associated compression, dorsal compression, alterations in ISI, new bone formation, and suspected clinical status. There was very good intraobserver agreement for ratings of disk degeneration, disk-associated compression, alterations in ISI, vertebral body abnormalities, and suspected clinical status. There was good intraobserver agreement for ratings of dorsal compression and new bone formation. Two of 21 clinically affected dogs were erroneously categorized as clinically normal, and 4 of 23 clinically normal dogs were erroneously categorized as clinically affected. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that variability exists among observers with regard to results of MRI in dogs with DAWS and that MRI could lead to false-positive and false-negative assessments.

Involvement of a chromomycin ABC transporter system in secretion of a deacetylated precursor during chromomycin biosynthesis.

Chromomycin A(3) is an antitumour antibiotic that acts by inhibiting transcription and replication of DNA. The producer micro-organism Streptomyces griseus subsp. griseus is highly resistant to chromomycin A(3) and to the structurally related compound mithramycin upon induction with chromomycin A(3). The biosynthetic gene cluster of chromomycin contains three genes involved in self-resistance to chromomycin in S. griseus: cmrA and cmrB encode a type I ATP-binding cassette (ABC) transporter, and cmrX encodes a UvrA-like protein of ABC excision nuclease systems. These genes are linked in the chromosome, together with a gene encoding a transcriptional repressor (cmmRII). Involvement of these genes in chromomycin resistance was determined through gene inactivation, and heterologous expression in Streptomyces albus. Inactivation of cmrX produced a chromomycin-sensitive low-producer strain, while inactivation of cmmRII generated a high-chromomycin-producer strain, which was resistant to chromomycin, and also to mithramycin. Expression of either cmrA and cmrB, or cmrX, in S. albus generated strains with low chromomycin resistance; it was therefore necessary to co-express the three genes to achieve high levels of resistance. However, the CmrAB ABC transporter conferred a high level of resistance to the biosynthesis intermediate 4A,4E-O-dideacetyl-chromomycin A(3). A model is proposed for the biosynthesis of, and self-resistance to, chromomycin A(3) in S. griseus subsp. griseus.

Lymphoma affecting the urinary bladder in three dogs and a cat.

Three dogs and one cat with lymphoma affecting the urinary bladder are reported and the findings on abdominal radiographs and ultrasound are described. Mural lesions representing lymphoma affecting the urinary bladder were identified ultrasonographically in all animals. The most common complications associated with urinary bladder lymphoma were hydronephrosis and hydroureter. In two patients contrast radiography was necessary to detect leakage of urine in the peritoneal and retroperitoneal space. The radiographic and ultrasonographic signs were similar to those reported with other urinary bladder neoplasms; hence urinary bladder lymphoma could not be distinguished from the more common urinary bladder neoplasms, such as transitional cell carcinoma. It is important to include lymphoma in the differential diagnosis of urinary bladder wall thickening and mural mass in dogs and cats.

The aureolic acid family of antitumor compounds: structure, mode of action, biosynthesis, and novel derivatives.

Members of the aureolic acid family are tricyclic polyketides with antitumor activity which are produced by different streptomycete species. These members are glycosylated compounds with two oligosaccharide chains of variable sugar length. They interact with the DNA minor groove in high-GC-content regions in a nonintercalative way and with a requirement for magnesium ions. Mithramycin and chromomycins are the most representative members of the family, mithramycin being used as a chemotherapeutic agent for the treatment of several cancer diseases. For chromomycin and durhamycin A, antiviral activity has also been reported. The biosynthesis gene clusters for mithramycin and chromomycin A(3) have been studied in detail by gene sequencing, insertional inactivation, and gene expression. Most of the biosynthetic intermediates in these pathways have been isolated and characterized. Some of these compounds showed an increase in antitumor activity in comparison with the parent compounds. A common step in the biosynthesis of all members of the family is the formation of the tetracyclic intermediate premithramycinone. Further biosynthetic steps (glycosylation, methylations, acylations) proceed through tetracyclic intermediates which are finally converted into tricyclic compounds by the action of a monooxygenase, a key event for the biological activity. Heterologous expression of biosynthetic genes from other aromatic polyketide pathways in the mithramycin producer (or some mutants) led to the isolation of novel hybrid compounds.

Deoxysugar transfer during chromomycin A3 biosynthesis in Streptomyces griseus subsp. griseus: new derivatives with antitumor activity.

Chromomycin A3 is an antitumor drug produced by Streptomyces griseus subsp. griseus. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-O-acetyl-D-oliose (sugar A) and 4-O-methyl-D-oliose (sugar B), and a trisaccharide of D-olivose (sugar C), D-olivose (sugar D), and 4-O-acetyl-L-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (cmmGI, cmmGII, cmmGIII, and cmmGIV), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-O-deacetyl-3A-O-acetyl-prechromomycin A4, and 3A-O-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two D-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A3 biosynthesis.

Tailoring modification of deoxysugars during biosynthesis of the antitumour drug chromomycin A by Streptomyces griseus ssp. griseus.

Chromomycin A3 is a member of the aureolic acid group family of antitumour drugs. Three tailoring modification steps occur during its biosynthesis affecting the sugar moieties: two O-acetylations and one O-methylation. The 4-O-methylation in the 4-O-methyl-D-oliose moiety of the disaccharide chain is catalysed by the cmmMIII gene product. Inactivation of this gene generated a chromomycin-non-producing mutant that accumulated three unmethylated derivatives containing all sugars but differing in the acylation pattern. Two of these compounds were shown to be substrates of the methyltransferase as determined by their bioconversion into chromomycin A2 and A3 after feeding these compounds to a Streptomyces albus strain expressing the cmmMIII gene. The same single membrane-bound enzyme, encoded by the cmmA gene, is responsible for both acetyl transfer reactions, which convert a relatively inactive compound into the bioactive chromomycin A3. Insertional inactivation of this gene resulted in a mutant accumulating a dideacetylated chromomycin A3 derivative. This compound, lacking both acetyl groups, was converted in a two-step reaction via the 4E-monoacetylated intermediate into chromomycin A3 when fed to cultures of S. albus expressing the cmmA gene. This acetylation step would occur as the last step in chromomycin biosynthesis, being a very important event for self-protection of the producing organism. It would convert a molecule with low biological activity into an active one, in a reaction catalysed by an enzyme that is predicted to be located in the cell membrane.

Biosynthesis of the antitumor chromomycin A3 in Streptomyces griseus: analysis of the gene cluster and rational design of novel chromomycin analogs.

The biosynthetic gene cluster of the aureolic acid type antitumor drug chromomycin A3 from S. griseus subsp. griseus has been identified and characterized. It spans 43 kb and contains 36 genes involved in polyketide biosynthesis and modification, deoxysugar biosynthesis and sugar transfer, pathway regulation and resistance. The organization of the cluster clearly differs from that of the closely related mithramycin. Involvement of the cluster in chromomycin A3 biosynthesis was demonstrated by disrupting the cmmWI gene encoding a polyketide reductase involved in side chain reduction. Three novel chromomycin derivatives were obtained, named chromomycin SK, chromomycin SA, and chromomycin SDK, which show antitumor activity and differ with respect to their 3-side chains. A pathway for the biosynthesis of chromomycin A3 and its deoxysugars is proposed.