Biomedical subjects

C Daly

Publications and source records attributed to C Daly.

At least 91 records · Page 5Linked to original sources

PubMed1992

A soluble form of Fc gamma RIII is present in human serum and other body fluids and is elevated at sites of inflammation.

We have developed a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) to measure the concentration of Fc gamma RIII in serum and other body fluids. This ELISA is based on the use of monoclonal antibody (MoAb) (3G8) to Fc gamma RIII and a rabbit antiserum against Fc gamma RIII. The lower limit of detection of this ELISA was 1.5 nmol/L. The concentration of soluble Fc gamma RIII in normal serum ranged from 7.3 to 75.9 nmol/L. Soluble Fc gamma RIII was also present in other normal biologic fluids such as saliva, urine, and seminal fluid, but at much lower concentrations than that found in serum. Rabbit anti-Fc gamma RIII immunoblotted polypeptides immunoprecipitated with MoAb 3G8. Fc gamma RIII immunoprecipitated from a neutrophil lysate migrated from 40 to 76 Kd, whereas Fc gamma RIII immunoprecipitated from serum from the same donor migrated from 40 to 66 Kd. The soluble form of Fc gamma RIII apparently was bound to serum IgG, because immunoprecipitation of soluble Fc gamma RIII by MoAb 3G8 coprecipitated polypeptides that were identified by goat antihuman IgG. Incubation of neutrophils in vitro at 4 degrees C and 37 degrees C showed that Fc gamma RIII was released after 30 minutes of incubation at 37 degrees C. To determine whether there was a correlation between the concentration of soluble Fc gamma RIII in biologic fluids and inflammatory diseases, we measured the concentration of Fc gamma RIII in the bronchoalveolar lavage fluid from patients with adult respiratory distress syndrome (ARDS) and in the synovial fluid from patients with various forms of arthritis. In ARDS, we found concentrations of soluble Fc gamma RIII that were five to seven times higher than that found in the bronchoalveolar lavage fluids from healthy adults. The concentration of soluble Fc gamma RIII in the synovial fluid from patients with rheumatoid arthritis ranged from 10 nmol/L to 28 mumol/L. These results suggest that activated neutrophils, such as those at sites of inflammation, may release Fc gamma RIII.

Antigens, Differentiation↗

PubMed1992

Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317.

The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCDO763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.

Amino Acid Sequence↗

PubMed1992

Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829.

Lactobacillus helveticus 1829 produced an antimicrobial agent, designated helveticin V-1829, that demonstrated antagonistic activity against closely-related species. The agent was excreted into MRS agar, and was present in the supernatant fluids from both overnight broth and clotted milk cultures. It was heat labile (inactivated by 50 degrees C for 30 min) and was stable over the pH range 2.5 to 6.5. Production of the substance was pH-dependent and maximum yields were obtained in MRS broth cultures maintained at pH 5.5. Helveticin V-1829 was partially purified following growth of the producing strain in a semi-defined MRS medium and precipitating the cell-free filtrate with ammonium sulphate to 30% saturation. The cleared supernatant fluid was then brought to 60% saturation and the resulting precipitate pelleted and dialysed in 0.3 mol/l phosphate buffer. The partially purified inhibitor was sensitive to several proteolytic enzymes, and it was bactericidal in its mode of action against indicator cells of Lact. helveticus 1844 and Lact. delbrueckii subsp. bulgaricus 1489, indicating that it was a bacteriocin. A DNA probe specific for the helveticin J structural gene failed to hybridize to total genomic DNA of Lact. helveticus 1829, indicating that helveticin V-1829 is not significantly related to helveticin J.

Bacteriocins↗

PubMed1992

Controlled Integration into the Lactococcus Chromosome of the pCI829-Encoded Abortive Infection Gene from Lactococcus lactis subsp. lactis UC811.

The phage insensitivity gene of lactococcal plasmid pCI829 which encodes an abortive infection defense mechanism (Abi) was inserted into the Lactococcus lactis subsp. lactis CH919 chromosome by utilizing the integration plasmid pCI194, which contains 4.2 kb of homology with the conjugative transposon Tn919. Chloramphenicol-resistant transformants expressed phage insensitivity to the prolate-headed phage c2 and the small isometric-headed phage 712, and hybridization analysis indicated that transformants contained pCI194 integrated in single copy. The level of phage insensitivity expressed by the transformants was reduced from that observed when the abi gene was located on a replicating plasmid, as determined by plaque assay and burst size analysis. Amplification of the integrated structure after growth in increased concentrations of chloramphenicol resulted in an increase in the expression of phage insensitivity. Hybridization analysis revealed that while pCI194 was stably maintained in an integrated state over 100 generations in the absence of selective pressure, the ability to express phage insensitivity was lost. Hybridization analysis also revealed that DNA flanking the abi gene contains homology to the CH919 chromosome.

Journal Article↗

PubMed1992

Avascular necrosis of the femoral head post heart-transplantation and steroid dosage.

Avascular necrosis (avn) post heart-transplantation has been considered to be due to the high doses of steroids used to immunosuppress these patients in attempting to prevent transplant rejection. This study shows that avascular necrosis occurs even when low dose steroids regimes are used and demonstrates no significant correlation between steroid dosage and the development of avn. Patients with symptomatic avn benefit from early diagnosis and management of their condition in that the need for total joint arthroplasty can be prevented in many cases.

Adult↗

PubMed1992

The role of selection bias in comparing cesarean birth rates between physician and midwifery management.

OBJECTIVE: The midwifery service at our hospital has been observed to have a 2% cesarean birth rate consistently over a 10-year period. There are substantial differences in labor management style between the midwives and physicians. We sought to test the hypothesis that the low cesarean birth rate on the midwifery service was the result of patient selection bias. METHODS: A randomized blinded clinical trial was conducted in which 492 low-risk patients were assigned to either physician or midwifery management. The provider responsible for labor management was unable to determine group assignment. Patients in the midwifery group were managed by previously established protocols, and outcome was attributed to the midwives even if the patients subsequently required transfer to physician management. Route of delivery was the primary outcome measurement. Continuous variables were analyzed using Student t test and discrete variables using chi 2. RESULTS: There were no demographic differences between the groups, and the admission pelvic examinations were the same. The patients assigned to the midwifery group had a 2.1% cesarean birth rate, whereas those assigned to physician management had a 0.4% rate. The higher rate of operative vaginal deliveries in the physician group was statistically significant. There were no differences in neonatal outcomes. The physician-managed group had significantly more episiotomies and third- and fourth-degree extensions. CONCLUSIONS: The 2% cesarean birth rate observed on the midwifery service appeared to be the result of patient selection bias. A low cesarean birth rate can be achieved by either physician or midwifery management in a selected low-risk population.

Adult↗

PubMed1991

Molecular organization of the minimal replicon of novel, narrow-host-range, lactococcal plasmid pCI305.

Plasmid pCI305 is an 8.7-kb, narrow-host-range, cryptic plasmid originating from Lactococcus lactis subsp. lactis UC317. The nucleotide sequence of the pCI305 replication region was determined. A single open reading frame of 1158 bp was identified in the trans-active domain repB. The size of the predicted repB protein (46 kDa) is in close agreement with the size of the repB product visualized in vivo in Escherichia coli when repB was placed under control of the inducible phi T7 RNA polymerase promoter. In vivo substitution of the native repB promoter sequence with a Tn5-derived promoter sequence was demonstrated. repA, a 344-bp cis-acting region which is the probable pCI305 replication origin region, was noncoding, was AT-rich, and possessed a unique set of inverted and direct repeat sequences. No significant homology between repA or repB and other gram-positive replication regions was evident. Combined with the absence of a detectable single-stranded DNA intermediate during replication, these results indicate that the pCI305 replication region differs markedly from most gram-positive replicons examined to date. The presence on other lactococcal plasmids of replication regions related to that of pCI305 was demonstrated.

Amino Acid Sequence↗

PubMed1991

Cloning and characterization of the determinant for abortive infection of bacteriophage from lactococcal plasmid pCI829.

The genetic determinant for abortive infection of bacteriophage (Abi) from the lactococcal plasmid pCI829 was cloned on a 6.2 kb StuI fragment in Escherichia coli using the shuttle vector pSA3. In Lactococcus lactis subsp. lactis MG1363Sm the resulting recombinant plasmid pCI816 conferred complete insensitivity to the small isometric-headed phage 712 and a reduced plaque size in the case of the prolate-headed phage c2. The determinant was further localized by subcloning and nuclease Bal31 deletion analysis; approximately 2.0 kb of DNA was essential for the expression of the Abi+ phenotype. Nucleotide sequence analysis of this region revealed a putative open reading frame of 1887 base pairs preceded by a putative promotor sequence and ribosome-binding site which exhibited similarity to consensus E. coli and Bacillus subtilis transcription/translation signals. Hybridization experiments indicated that this region was not homologous to the abi determinant from the phenotypically similar lactococcal plasmid pCI750.

Amino Acid Sequence↗

PubMed1991

Chromosomal integration of plasmid DNA by homologous recombination in Enterococcus faecalis and Lactococcus lactis subsp. lactis hosts harboring Tn919.

Integration of pCI192, a pBR322-derived vector plasmid containing homology to the chromosomally located conjugative transposon Tn919 was observed in two strains that harbor Tn919, namely, Enterococcus faecalis GF590 and Lactococcus lactis subsp. lactis CH919. Hybridization analysis indicated that single-copy integration of the plasmid had occurred at low frequency. The Tn919::plasmid structure was conjugated from an E. faecalis donor to a L. lactis recipient, although at lower frequencies than was Tn919. Segregation of the tetracycline and chloramphenicol resistance markers during conjugation was observed. The integration strategy described allows for DNA manipulations to be performed in an easily manipulated model host strain with the subsequent transfer of integrated structures by conjugation to any strain capable of receiving Tn919. The results indicate that homologous recombination events may be used to introduce plasmid-encoded genes to the lactococcal chromosome.

Chromosomes, Bacterial↗

PubMed1990

Integration and excision of plasmid DNA in Lactococcus lactis subsp. lactis.

The capacity of the 75-kb lactose-proteinase plasmid pCI301 from Lactococcus lactis subsp. lactis UC317 to recombine with the lactococcal chromosome was examined. Low-frequency integration of pCI301 sequences was detected following protoplast transformation of strain MG136Sm with total plasmid DNA from strain UC317. Excision of integrated sequences was subsequently observed at a low level. Excised sequences were rescued through recombination with and mobilization by the conjugative enterococcal plasmid pAMB1. Transconjugants harboring novel recombinant pCI301::pAMB1 plasmids, both pAMB1 and a pCI301 derivative, and pAMB1 only were isolated. The latter represents a class of transconjugant in which an elevated level of reintegration of pCI301 DNA in the recipient chromosome has occurred.

Chromosomes, Bacterial↗

PubMed1990

pAMbeta1-Associated Mobilization of Proteinase Plasmids from Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205.

A combination of plasmid curing and DNA-DNA hybridization data facilitated the identification of proteinase plasmids of 75 (pCI301) and 35 kilobases (pCI203) in the multi-plasmid-containing strains Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205, respectively. Both plasmids were transferred by conjugation to a plasmid-free background only after introduction of the conjugative streptococcal plasmid, pAMbeta1. All Prt transconjugants from matings involving either donor contained enlarged recombinant Prt plasmids. UC317-derived transconjugants were separable into different classes based on the presence of differently sized cointegrate plasmids and on segregation of the pCI301-derived Lac and Prt markers. All UC205-derived transconjugants harbored a single enlarged plasmid that was a cointegrate between pCI203 and pAMbeta1. The identification of prt genes on pCI301 and pCI203 derivatives was achieved by a combination of restriction enzyme and hybridization analyses.

Journal Article↗

PubMed1990

Identification of the Minimal Replicon of Lactococcus lactis subsp. lactis UC317 Plasmid pCI305.

Replication functions of the stable, cryptic 8.7-kilobase (kb) plasmid pCI305 from multi-plasmid-containing Lactococcus lactis subsp. lactis UC317 were studied. Analysis of this replicon was facilitated by the construction of replication probe vectors that consisted of the pBR322 replication region, a pUC18-derived multiple cloning site, and either the cat gene of pC194 (pCI341; 3.1 kb) or the erm gene of pAMbeta1 (pCI3330; 4.0 kb). Plasmid pCI305 was introduced into plasmid-free L. lactis subsp. lactis MG1363Sm, a streptomycin-resistant derivative of MG1363, by a transformation procedure with the 75-kb lactose-proteinase plasmid pCI301 of UC317 as a marker plasmid. A combination of transposon Tn5 mutagenesis and subcloning in pCI341 and pCI3330 with individual Tn5 insertions around the replication region facilitated the identification of a 1.6-kb minimal replicon on pCI305. This region was separable into two domains: (i) a 1.3-kb region (repB) encoding a trans-acting function (in vitro transcription-translation studies suggested the involvement of a 48-kilodalton protein); and (ii) a 0.3-kb region (repA) sufficient to direct replication when provided with repB in trans and thus probably containing the origin of replication. Lactococcus-Escherichia coli shuttle vectors based on the pCI305 replication region were constructed.

Journal Article↗

PubMed1990

High-frequency, site-specific recombination between lactococcal and pAM beta 1 plasmid DNAs.

In vivo recombination events involving the 75-kilobase lactose proteinase plasmid pCI301 of Lactococcus lactis subsp. lactis UC317 and the conjugative enterococcal plasmid pAM beta 1 were analyzed. A fragment, identified as containing the pCI301 recombination site, mediated greatly elevated levels of mobilization and recombination with pAM beta 1 when cloned in a nonmobilizable L. lactis-Escherichia coli shuttle vector. This latter recombination event was site and orientation specific on both plasmids. Recombination on pAM beta 1 was within the region associated with plasmid replication, but no effect on pAM beta 1 replication functions was detected. Resolution of recombinant plasmids generated derivatives indistinguishable from the parental plasmids.

Conjugation, Genetic↗

PubMed1990

Cellular transport and accumulation of thiotepa.

Because the transport and accumulation of N,N',N''-triethylenethiophosphoramide (thiotepa) by cells has not been characterized, these processes were investigated with [14C]thiotepa and cultured L1210 or freshly obtained human or avian RBCs. The octanol: phosphate-buffered saline (PBS) partition coefficient of thiotepa was 2.4 +/- 0.1 (n = 8). With this value, the permeability coefficient (Ps) for thiotepa was estimated to be between 2.8 x 10(-4) and 1.81 x 10(-3) cm/sec, and the half-life of accumulation of thiotepa by L1210 cells was estimated to be 0.063 to 0.40 seconds. Thiotepa accumulation by cells was measured after incubation of cells with [14C]thiotepa and subsequent harvesting of cells by centrifugation through silicone fluid. Thiotepa accumulation by L1210 cells was biphasic. The initial phase was rapid and essentially complete by 10 seconds. The amount of cell-associated 14C increased linearly with increasing extracellular concentrations of thiotepa or with increasing size of the cell pellet. The absolute amount of cell-associated 14C was consistent with that expected if the [14C]thiotepa had been evenly distributed in the incubation medium and a volume equal to that of the cell pellet had been sampled and counted. This rapid phase of thiotepa accumulation was not slowed when cells were incubated on ice. The second phase of [14C]thiotepa accumulation occurred at a rate much slower than that of the initial phase. This slower phase of drug accumulation was linear for at least 5 hours. The rate of 14C accumulation increased progressively over a range of extracellular thiotepa concentrations from 5 to 100 nmol/mL and could not be saturated under acceptable tissue culture conditions. The slower rate of 14C accumulation was ablated by incubating cells on ice and was reduced by 30% to 50% in the presence of 1mM of sodium azide or 2,4-dinitrophenol. The slow rate of accumulation of 14C reflected summation of a relatively stable or constant amount of exchangeable 14C and an amount of nonexchangeable 14C that increased linearly from almost undetectable levels at the start of the experiment to amounts equal to 64 +/- 11% of total cellular radioactivity after 5 hours. The initial association of [14C]thiotepa with both human and avian RBCs was also very rapid. Avian RBCs also exhibited a slow rate of 14C accumulation that was linear for at least 5 hours but that was 15% to 20% that of L1210 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Traditional birth attendants in the Gambia.

In developing countries traditional birth attendants help at 60-70% of births, despite rapidly expanding "Western-style" health services. Recognising the potential of this established service, the World Health Organisation has been involved in a programme of training and supervision of traditional birth attendants (TBAs) in developing countries since 1973. An evaluation of training programmes in 1986 showed considerable success with improved antenatal care, decreased neonatal tetanus, increased recognition and transfer to hospital of complications of pregnancy and delivery and encouraging improvements in many other indices of improved management. This short paper describes the health services available in a developing country, The Gambia, in West Africa, and reports an interview with a trained traditional birth attendant in a rural area of the country.

Gambia↗

PubMed1989

Cellular transport and accumulation of thiotepa in murine, human, and avian cells.

Because the transport and accumulation of thiotepa by cells has not been characterized, these process were investigated with [14C]thiotepa and cultured L1210 or freshly obtained human or avian RBC. The octanol:phosphate buffered saline partition coefficient of thiotepa was 2.4 +/- 0.1 (n = 8). With this value, the permeability coefficient (P) for thiotepa was estimated to be between 2.8 X 10(-4) and 1.81 X 10(-3) cm/s and the half-life of accumulation of thiotepa by L1210 cells was estimated to be 0.063-0.40 s. Thiotepa accumulation by cells was measured after incubation of cell with [14C]thiotepa and subsequent harvesting of cells by centrifugation through silicone fluid. Thiotepa accumulation by L1210 cells was biphasic. The initial phase was rapid essentially complete by 10 s. The amount of cell-associated 14C increased linearly with increasing extracellular concentrations of thiotepa or with increasing size of the cell pellet. The absolute amount of cell-associated 14C was consistent with that expected if the [14C]thiotepa had been evenly distributed in the incubation medium and a volume equal to that of the cell pellet had been sampled and counted. This rapid phase of thiotepa accumulation was not slowed when cells were incubated on ice. The second phase of [14C]thiotepa accumulation occurred at a rate much slower than that of the initial phase. This slower phase of drug accumulation was linear for at least 5 h. The rate of 14C accumulation increased progressively over a range of extracellular thiotepa concentrations between 5 and 100 nmol/ml and could not be saturated under acceptable tissue culture conditions. The slower rate of 14C accumulation was ablated by incubation cells on ice and was reduced by 30-50% in the presence of 1 mM sodium azide or 2,4-dinitrophenol. The slow rate of accumulation of 14C reflected summation of a relatively stable or constant amount of exchangeable 14C an an amount of nonexchangeable 14C which increased linearly from almost undetectable levels at the start of the experiment to amounts approximately equal to those of exchangeable radioactivity after 5 h. The initial association of [14C]thiotepa with both human and avian RBCs was also very rapid. Avian RBCs also exhibited a slow rate of 14C accumulation which was linear for at least 5 h which was 15-20% that of L1210 cells. Human RBCs did not exhibit a slower rate of 14C accumulation and essentially all of the 14C associated with human RBCs was exchangeable for the 5 h duration of the experiment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

DNA-DNA homology among lactose- and sucrose-fermenting transconjugants from Lactococcus lactis strains exhibiting reduced bacteriophage sensitivity.

DNA-DNA homology between a reduced bacteriophage sensitivity (Rbs+) probe and DNA from both Rbs+ and Rbs- Lactococcus lactis strains was examined. Homology was detected between the probe and five plasmids (pCI750, pCC34, pEB56, pNP2, and pJS88) isolated from lactose-positive Rbs+ transconjugants and between the probe and genomic DNA of a sucrose-positive Rbs+ transconjugant. Additionally, hybridizations conducted between the probe and plasmids reported to encode abortive bacteriophage infection indicated homology with pTR2030 but not with pBF61 and pGBK17. The results suggest that a common genetic determinant(s) may be present in a variety of lactococcal plasmids coding for Rbs+.

Bacteriophages↗

PubMed1989

[Physiologic reactions during the slaughter of cattle and sheep with and without stunning].

In a first trial the cortical activity of cattle and sheep was tested using only an electrocorticogram (ECoG). The results showed a shorter phase after sticking or the ritual slaughter cut respectively, until the ECoG disappears in stunned animals. However, considering the time between stunning and the cervical state, the interval until disappearance of the ECoG was prolonged in the stunned animals. The observed time differences, however, are only a tendency and not statistically confirmed. Insofar, concerning animal protection, the different slaughter methods could be regarded equivalent. A second trial was designed in a manner to allow a more exact interpretation of the ECoGs and was completed by measuring visually and somatosensorically evoked potentials. Additionally, in contrast to the first trial, only adult cattle were used here. The results revealed shorter intervals until disappearance of cortical activities when using captive-bolt stunning. Also the variance was much lower in this trial than after ritual slaughter. The mean of the time differences was relatively low (5.5 seconds). It was especially remarkable that after captivebolt stunning absolutely no evoked potentials could be registrated, whereas these potentials lasted for 77 seconds (somatosensorically evoked potentials) and 55 seconds respectively (visually evoked potentials) after the ritual slaughter cut. Thus, after ritual slaughter a nervous conduction was measured up to 126 seconds in the extreme cases. However, from the results obtained it can not be concluded whether or not pain sensitivity occurred in the animals.

Abattoirs↗