Prognostic value of power doppler ultrasonography for equine superficial digital flexor tendon injury in thoroughbred racehorses.

The potential value of hypervascularity detected with power Doppler ultrasonography (PDU) within equine superficial digital flexor tendon (SDFT) as a prognostic factor of SDFT injury is not clear. The purpose of this study was to test the hypothesis that hypervascularity within SDFT is one of the risk factors for subsequent severe SDFT injury and to evaluate the prognostic value. A prospective cohort study of 97 Thoroughbred racehorses without any clinical signs of SDFT injury was conducted. Six variables of age, body weight, sex, the cross-sectional area of SDFT, PDU signal within SDFT and experience of steeplechase were assessed for the possibility of risk factors of subsequent SDFT injury in follow-up period of 1 year. Multivariable logistic regression analyses were used for assessment of the odds ratios (ORs) and 95 % confidence intervals (CIs) of SDFT injury. Multivariable logistic regression analysis revealed that the PDU signal within SDFT was a risk factor for the development of SDFT injury in follow-up period (P = 0.017). The adjusted OR of SDFT injury was significantly higher in PDU positive group than in PDU negative group (OR 3.17, 95 % CIs 1.20-8.35). Although further studies are required, these results would be useful for early detection and/or prevention of development for clinical severe SDFT injury.

The C. elegans unc-18 gene encodes a protein expressed in motor neurons.

The C. elegans unc-18 gene is required to maintain normal acetylcholine levels. We determined the complete structure of an unc-18 cDNA that encodes a protein of 591 highly charged and hydrophilic amino acids. The protein shows sequence similarity with elements of the secretory pathway in the yeast S. cerevisiae. Antibodies raised against a portion of the unc-18-encoded protein (UNC-18) detected a 68 kd soluble antigen on immunoblots and intensely stained all vertical cord motor neurons in situ. These findings suggest that UNC-18 participates in the axonal transport system and influences the acetylcholine flow in motor neurons.

A retrospective cohort study investigating risk factors for the failure of Thoroughbred racehorses to return to racing after superficial digital flexor tendon injury.

A retrospective cohort study was conducted to investigate risk factors for the failure of Thoroughbred racehorses to return to racing after an injury of the superficial digital flexor tendon (SDFT). Successful return was defined as the completion of five or more races after SDFT injury. The official Japan Racing Association (JRA) medical records of racehorses with a core-type SDFT injury were reviewed for clinical variables related to the characteristics of the horse and the severity of SDFT injuries at the time of diagnosis. Data on racing outcomes were obtained from the official JRA racing database. Risk factors were screened using univariable logistic regression and subsequent multivariable model building. Forty-nine of 346 (14.2%) horses successfully returned to racing after SDFT injuries. Multivariable model building revealed that an increase in the total number of injured zones (defined as the total number of zones in which the injured hypoechoic area was observed at the time of ultrasonographic diagnosis of SDFT injury) was associated with an increased risk of failure to return to racing after SDFT injury. Horse characteristics, such as age, body mass and sex, were not associated with a successful return to racing. In the rehabilitation of cases with larger (longer) lesions, more effective and careful medical management may be needed for an improvement in the athletic outcomes.

Comparison of alfaxalone, ketamine and thiopental for anaesthetic induction and recovery in Thoroughbred horses premedicated with medetomidine and midazolam.

REASONS FOR PERFORMING STUDY: There is limited information on clinical use of the new injectable anaesthetic agent alfaxalone in Thoroughbred horses. OBJECTIVES: To compare anaesthetic induction and recovery characteristics and cardiopulmonary responses between alfaxalone, ketamine and thiopental in Thoroughbred horses premedicated with medetomidine and midazolam. STUDY DESIGN: Randomised blinded experimental cross-over study. METHODS: Six Thoroughbred horses were anaesthetised 3 times with alfaxalone 1 mg/kg bwt, ketamine 2.5 mg/kg bwt or thiopental 4 mg/kg bwt after premedication with medetomidine 6 µg/kg bwt and midazolam 20 µg/kg bwt. Qualities of anaesthetic induction and recovery were scored on a scale of 1 (poor) to 5 (excellent). Induction time and recovery time were recorded. Cardiopulmonary values (heart rate, respiratory rate, arterial blood pressures, and arterial blood gases) were recorded throughout anaesthesia. Data were analysed with nonparametric methods. RESULTS: The anaesthetic induction (P = 0.2) and recovery (P = 0.1) quality scores (median, range) were not different amongst protocols and were 4.0, 3-5; 5.0, 4-5; 4.5, 3-5; and 4.5, 3-5; 3.5, 2-5; 4.0, 2-5 for alfaxalone, ketamine and thiopental, respectively. Induction time for ketamine (67, 53-89 s) was significantly longer than that for alfaxalone (49, 40-51 s, P = 0.01) and thiopental (48, 43-50 s, P = 0.01). Time to standing for alfaxalone (44, 40-63 min, P = 0.01) and thiopental (39, 30-58 min, P = 0.01) was significantly longer than that for ketamine (25, 18-26 min). Cardiovascular values were maintained within the clinically acceptable level throughout anaesthesia. Respiratory rate significantly decreased during anaesthesia for all 3 drugs; however, spontaneous breathing did not disappear, and PaCO2 values were maintained at approximately 50 mmHg. CONCLUSIONS: All 3 drugs showed similar effects in relation to anaesthetic induction and recovery qualities and cardiopulmonary responses. However, alfaxalone and thiopental prolonged recovery time compared with ketamine.

Replication of bacteriophage G13 DNA in dna mutants of Escherichia coli.

Host functions required for replication of microvirid phage G13 DNA were investigated in vivo, using thermosensitive dna mutants of Escherichia coli. In dna+ bacteria, conversion of viral single-stranded DNA into double-stranded replicative form (stage I synthesis) was resistant to 150 microgram/ml of chloramphenicol or 200 microgram/ml of rifampicin. Although multiplication of G13 phage was severely inhibited at 42--43 degrees C even in dna+ host, considerable amount of parental replicative form was synthesized at 43 degrees C in dna+, dnaA or dnaE bacteria. In dnaB and dnaG mutants, however, synthesis of parental replicative form was severely inhibited at the restrictive temperature. Interestingly enough, stage I replication of G13 DNA was, unlike that of phiX174, dependent on host dnaC(D) function. Moreover, the stage I synthesis of G13 DNA in dnaZ was thermosensitive in nutrient broth but not in Tris/casamino acids/glucose medium. In contrast with the stage I replication, synthesis of G13 progeny replicative form was remarkably thermosensitive even in dna+ or dnA cells.

Function and structure of microvirid phage alpha 3 genome. II. Isolation and properties of various mutants of alpha 3.

Various mutants were isolated from a microvirid (isometric single-stranded DNA) phage alpha 3, by mutagenesis with hydroxylamine or nitrous acid. They were divided into eight complementation groups, and mainly by genetic crosses the gene alignment was determined as -A-B-C'-D-J'-F-G-H-. Except for groups C' and J', each defective gene product was clearly discerned in electropherograms of proteins extracted from the phage-infected suppressor-negative (Su-) Escherichia coli. Only gene A mutants abolished synthesis of the progeny replicative-form DNA (RF), whereas mutants belonging to groups B, C', D, E, F and J' affected RF replication at late stage, as well as synthesis of the single-stranded DNA (SS). Additional properties of several mutants are also discussed.

Synthesis of alpha3 phage DNA in replication mutants of Escherichia coli.

Host functions for DNA replication of bacteriophage alpha3, a representative of group A microvirid phages, were studied using dna and rep mutants of Escherichia coli. In dna+ cells, conversion of phage alpha3 single-stranded DNA (SS) into the double-stranded replicative form (RF) was insensitive to 30--150 microgram/ml of chloramphenicol, 200 microgram/ml of rifampicin, 50 microgram/ml of nalidixic acid, or 200 microgram/ml of novobiocin. At 43 degrees C, synthesis of the parental RF was inhibited in dnaG and dnaZ mutants, but not in dnaE and rep strains. Replication of phage alpha3 progeny RF was prevented by 50 microgram/ml of mitomycin C (in hcr+ bacteria), 50 microgram/ml of nalidixic acid or 200 microgram/ml of novoviocin, but neither by 30 microgram/ml of chloramphenicol nor by 200 microgram/ml of rifampicin. Besides dnaG and dnaZ gene products, dnaE and rep functions were essential for progeny RF synthesis. Host factor dependence of alpha3 was relatively simple and, in contrast with phages phiX174 and G4, alpha3 did not require dnaB and dnaC(D) activities.

Function and structure of microvirid phage alpha 3 genome. DNA sequence of H gene and properties of missense H mutant.

The nucleotide sequence of wild-type alpha 3 H gene and its surrounding region was determined and compared with those of phi X174 and G4. The corresponding DNA regions in double mutants amJH22, amJH69 and amJH76 were also sequenced and their missense mutation sites located. A phage strain missH22 having a single missense mutation in gene H was constructed by replacing the J region of amJH22 in vitro with the wild-type DNA. Like amJH22, the missense mutant coded for H protein with aberrant electrophoretic mobility, but formed normal plaques on suppressor-deficient Escherichia coli. Heat stability, plating efficiency on certain hosts and rate of eclipse were higher in strain missH22 than in wild-type phage.

Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative form in dna mutants of Escherichia coli.

Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.

Properties of the bacteriophage alpha 3 mutants with deletion and/or insertion in the complementary strand origin.

Bacteriophage alpha 3 origin of complementary strand DNA synthesis (-ori) contains two potential secondary loop structures (I and II), which have been implicated in direct recognition sites for host Escherichia coli dnaG protein. We have introduced nucleotide deletion or insertion within the -ori region, by nuclease digestion and polymerase treatment of alpha 3 replicative form DNA. Deletion mutants (delo) showing the following in vivo properties were isolated: minute plaque size, longer latent period, and reduction both in phage yield and viral DNA synthesis as compared with the wild-type phage. In addition, several strains among the delo mutants did not grow on host E. coli dnaB cells at 42 degrees C. These results suggest that certain type of deletion in the -ori region converts simple priming of the complementary strand synthesis to a more complicated phi X174-type initiation that depends on the primosome, rather than on dnaG protein alone.

Mutational analysis of the bacteriophage alpha 3 origin of complementary DNA synthesis: in vivo properties of mutants.

Bacteriophage alpha 3 origin of complementary strand DNA synthesis contains two potential secondary loop structures, I and II, which have been implicated in direct recognition sites for host Escherichia coli dnaG protein. To elucidate the function of the hairpin loops, we have introduced point mutations within the stem of the hairpin II so as to disturb its base-pairings. A mutant, oriAA, which had two point mutations in the region, formed minute plaques on E. coli host cells and its mean burst size at 37 degrees C was about 50, whereas that of wild-type was 250. In addition, the growth of oriAA at 42 degrees C was thermosensitive and the burst size was reduced to 5. From the oriAA, a revertant-like phage oriGA occurred spontaneously with a high-frequency of about 2.10(-2). It retained one point mutation and the plaque size and phage yield were nearly same as those of wild-type. These results are discussed with respect to the role of secondary structure as well as specific nucleotide sequence in the recognition site for the dnaG protein.

Comparative studies on the minus origin mutants of Escherichia coli spherical single-stranded DNA phages.

The minus origins for complementary strand DNA synthesis (-ori) of Escherichia coli spherical single-stranded DNA (microvirid) phages G4, phi K, alpha 3, and St-1 closely resemble each other in DNA structure and contain two potential secondary hairpin loops (I and II) that have been implicated as direct recognition sites for host E. coli dnaG protein (primase). We introduced mutations (deletion or insertion) within the -ori regions of phi K and G4 by the nuclease digestion method. Mutants thus constructed produced minute plaques, showed thermosensitivity, and they remarkably reduced the phage yield and rate of viral DNA synthesis. Deletions in the phi K mutants (dTa) were ranging from 1 nucleotide (nt) to 102 nt centered at the hairpin II; a dTa8 mutant was entirely lacking in the two hairpins besides the starting point for primer RNA synthesis. On the other hand, the G4 mutants (dSa) had deletions centered at hairpin I; two mutants dSa35 and dXN completely lost the hairpin I and the primer RNA starting point. In addition, progeny phage populations of several phi K and G4 mutants contained revertant-like phages. DNA sequencing analysis revealed that these secondary phages had been generated by spontaneous DNA rearrangement with additional insertion or deletion near the parental mutation sites, via an unknown recA-independent pathway.

Nucleotide sequence of the genome of the bacteriophage alpha 3: interrelationship of the genome structure and the gene products with those of the phages, phi X174, G4 and phi K.

The complete nucleotide sequence of the genome of the circular single-stranded DNA (isometric) phage alpha 3 has been determined and compared with that of the related phages phi X174 and G4. The alpha 3 genome consists of 6087 nucleotides, which is 701 nucleotides longer than the nucleotide sequence of the phi X174 genome and 510 nucleotides more than that of the G4 genome. The results demonstrated that the three phage species have 11 homologous genes (A, A*, B, C, K, D, E, J, F, G and H), the order of which is fundamentally identical, suggesting that they have evolved from a common ancestor. The sequence of some genes and untranslated intergenic regions, however, differs significantly from phage to phage: for example, the degree of amino acid sequence homology of the gene product is averaged at 47.7% between alpha 3 and phi X174 and 46.9% between alpha 3 and G4, and alpha 3 has a remarkable longer intergenic region composed of 758 nucleotides between the genes H and A compared with the counterparts of phi X174 and G4. Meanwhile, in vivo experiments of genetic complementation showed that alpha 3 can use none of the gene products of phi X174 and G4, whereas the related phage phi K can rescue alpha 3 nonsense mutants of the genes B, C, D and J. These sequencing and in vivo rescue results indicated that alpha 3 is closely related to phi K, but distantly remote from phi X174 or G4, and supported an evolutional hypothesis which has been so far proposed that the isometric phages are classified into three main groups: the generic representatives are phi X174, G4 and alpha 3.

Determination of the single strand origin of Shigella sonnei plasmid pKYM.

The Shigella sonnei plasmid pKYM replicates by a rolling-circle mechanism in Escherichia coli. A 571 nucleotides HincII restriction fragment of the pKYM DNA harbors two potential hairpin loops (I and II). We cloned the fragment into a -ori defective M13 vector phage, M13 delta lac183. The chimera phage, MDKY5, showed a larger plaque size, and increased phage yield and rate of progeny replicative form DNA (RF) synthesis. Rifampicin reduced rate of conversion of the single- to double-stranded RF DNA. In addition, we introduced nucleotide deletions within the cloned pKYM DNA, by Bal31 nuclease digestion. Each of the deletion mutants thus constructed was lacking in a sequence containing the hairpin loops and formed smaller plaques. The in vivo analyses revealed that a 136 nucleotides sequence containing the two hairpins I and II is the pKYM minus origin for complementary strand synthesis (single strand origin, referred to as SSO) and harbors a recognition site(s) by host E. coli RNA polymerase, for primer RNA synthesis. Moreover, we found a 24 nt sequence, upstream of the SSO domain having 83% homology to the recombination site A (RSA) which functions in plasmid sitespecific recombination and/or transfer.

Purification and biochemical properties of an N-hydroxyarylamine O-acetyltransferase from Escherichia coli.

The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS-PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.

Possible finger structure in gene A protein of Microviridae.

Microvirid phages alpha 3 and phi K encode for A protein which functions in initiation of the viral DNA synthesis. By nucleotide sequencing analysis, we have found that each gene A protein has 'finger motif structure' which conserves two cysteine and histidine residues similar to the consensus sequence deduced from more than thirty finger motifs reported from many eukaryotic regulatory proteins. In closely related phages, phi X174 and G4, we have detected the same structures in their gene A proteins also.

Cloning and characterization of the gene encoding mouse osteoclast differentiation factor.

Osteoclast differentiation factor (ODF), a ligand for osteoclastogenesis inhibitory factor (OCIF)/ osteoprotegerin (OPG), is a member of the membrane-associated tumor necrosis factor (TNF) family and induces osteoclast-like cell formation in vitro. In the present study, mouse ODF genomic clones were isolated and sequenced to determine their gene structure. The mouse ODF gene is a single copy gene consisting of five exons and spans approximately 40kb of the mouse genome. The first exon encodes the intracellular and transmembrane domains. The extracellular region of ODF containing the TNF homologous domain is encoded by exons 1 through 5. The translation-termination codon and six polyadenylation signal residues are present in exon 5. A major transcription-initiation site is present 143 nucleotides upstream of the initiation-ATG codon. This genomic organization is similar to that of other members of the TNF family, especially the CD40 ligand.

Cloning, sequence analysis, and expression of the genes encoding lytic functions of Bacteriophage phi g1e.

The lysis genes of a Lactobacillus phage phi g1e were cloned, sequenced, and expressed in Escherichia coli. Nucleotide sequencing of a 3813-bp phi g1e DNA revealed five successive open reading frames (ORF), Rorf50, Rorf118, hol, and lys and Rorf175, in the same DNA strand. By comparative analysis of the DNA sequence, the putative hol product (holin) has an estimated molecular weight is 14.2 kDa, and contains two potential transmembrane helices and highly charged N- and C-termini, resembling predicted holins (which are thought to be a cytoplasmic membrane-disrupting protein) encoded by other phages such as mv1 from Lactobacillus bulgaricus, phi adh from Lactobacillus gasseri, as well as monocins from Listeria. On the other hand, the putative phi g1e lys product (lysin) of 48.4 kDa shows significant similarity with presumed muramidase, known as a cell wall peptidoglycandegrading enzyme, encoded by the Lactobacillus phage mv1 and phi adh, the Lactococcus lactis phage phi LC3, and the Streptococcus pneumoniae phages Cp-1, Cp-7 and Cp-9. When expressed in E. coli, the phi g1e lysin and/or holin decreased the cell turbidity significantly, suggesting that the phi g1e hol-lys system is involved in cytolytic process.

The genetic switch for the regulatory pathway of Lactobacillus plantarum phage (phi)g1e: characterization of the promoter P(L), the repressor gene cpg, and the cpg-encoded protein Cpg in Escherichia coli.

The structural and functional features of the approximately 530 bp P(L)/Gb5-Gb6-cpg-Gb7 region (P(L) overlaps Gb5) for the lysogenic pathway of L. plantarum phage (phi)gle were investigated using the cat gene of E. coli plasmid pKK232-8 as a reporter. In E. coli XL1-Blue, a recombinant plasmid pKPL2 (cat under P(L)/Gb5-Gb6) exhibited distinct CAT activity, whereas the activity of pKPLCP1 (cat under P(L)/Gb5-Gb6-cpg) was only marginal. When pKPL2 was coexistent with a compatible derivative of plasmid pACYC177 carrying P(L)/Gb5-Gb6-cpg, the CAT activity was declined to the level of pKPLCP1. On the other hand, the cpg-encoded protein Cpg was overproduced in E. coli under P(T7). The molecular mass of the purified Cpg (14.5 kDa on a SDS gel) corresponded well with that (15.1 kDa) predicted from the DNA sequence. Gel-shift and footprinting assays demonstrated that Cpg selectively binds to about 25 bp bases centered around the GATAC-box (from 1 to 7). Moreover, protein crosslinking experiments using glutaraldehyde showed that Cpg most likely functions as a dimeric form. Thus, the present results indicate that Cpg probably represses P(L) through binding to the operator GATAC-box(es), and the P(L)/cpg region might participate in the lysogenic pathway.

Purification and DNA-binding properties of the integrase protein Int encoded by Lactobacillus plantarum phage.

The Lactobacillus plantarum temperate phage phi g1e (42,259 bp) encodes an integrase gene int linked to a phage attachment site attP (Kakikawa et al., 1997). To investigate phi g1e recombination, the integrase protein Int was overproduced in Escherichia coli under the T7 promoter, and purified. The Int protein had an apparent molecular mass of 42.0 kDa, corresponding well with that (45.5 kDa) predicted from the DNA sequence. Amino-acid sequencing revealed that the N-terminal 20 amino-acids of the purified Int protein completely coincided with those deduced from the DNA sequence, although deficient in the first methionine. Gel mobility-shift assays demonstrated that Int bound specifically to the attP region. In addition, footprinting analysis showed that Int protected about 35 bases, containing the 24-bp core domain at attP, from DNase I attack. These results are indicative of site-specific interaction of Int with the attP site, the reaction prerequisite for integration and excision of the phi g1e genome into and/or out of the host chromosome.