The role of the operating department practitioner on board Role 2 Afloat.

The Role 2 Afloat (R2A) is the Royal Navy (RN)'s Damage Control Resuscitation (DCR), including Damage Control Surgery, capability at sea. There are currently three operating department practitioners (ODP) in the deployed team. This article describes the role of the ODP in this team and the training which is required to fulfil this role.

The role of the operating department practitioner on board Role 2 Afloat.

The Role 2 Afloat (R2A) is the Royal Navy (RN)'s Damage Control Resuscitation (DCR), including Damage Control Surgery, capability at sea. There are currently three operating department practitioners (ODP) in the deployed team. This article describes the role of the ODP in this team and the training which is required to fulfil this role.

An in vitro comparison of the bactericidal efficacy of lethal photosensitization or sodium hyphochlorite irrigation on Streptococcus intermedius biofilms in root canals.

AIM: To compare the bacterial killing of Streptococcus intermedius biofilms in root canals using lethal photosensitization with various combinations of photosensitizer concentration and laser light dose or 3% sodium hypochlorite (NaOCl) irrigation. METHODOLOGY: Extracted teeth (n = 35) with single canals were selected and the canals prepared to apical size 25 with a 10% taper. The teeth were autoclaved and the canals inoculated with Streptococcus intermedius in brain heart infusion broth and were incubated for 48 h to allow a biofilm to form. The teeth were then subjected to 3% NaOCl irrigation (n = 4) or lethal photosensitization using combinations of a range of toluidine blue O (TBO) photosensitizer concentrations (12.5, 25, 50, 100 microgram/mL-1) and light doses (60, 90, 120, 300, 600 s equivalent to energy doses of 2.1-21 J) using a 35-mW helium-neon low power laser targeted at the access cavity (n = 4 for each combination). Controls consisted of laser light only (TBO = 0 microgram/mL-1) (n = 4), TBO only (light dose = 0 s) (n = 4), and no treatment (positive control n = 17). Following treatment the canal contents were sampled with sterile paper points, the sample was dispersed in transport medium, serially diluted and cultured on blood agar to determine the number of colony forming units (CFU). RESULTS: The combination of 100 microgram/mL-1 TBO and 600 s (21 J) of laser energy achieved maximum reduction in recovered viable bacteria (5 log10 CFU). TBO at low concentrations (< or =50 microgram/mL-1) was not bactericidal but treatment with 100 microgram/mL-1 TBO alone reduced recovered viable bacteria by 3 log10 CFU. Laser light alone had limited bactericidal effect. No viable bacteria were recovered following treatment with 3% NaOCl. CONCLUSIONS: The combined use of a photosensitizing agent and a low power laser directed at the access cavity was bactericidal to S. intermedius biofilms in root canals but was unable to achieve total kill, unlike 3% NaOCl.

Purification and properties of the uracil DNA glycosylase from Bloom's syndrome.

Bloom's syndrome uracil DNA glycosylase was highly purified from two non-transformed cell strains derived from individuals from different ethnic groups. Their properties were then compared to two different highly purified normal human uracil DNA glycosylases. A molecular mass of 37 kDa was observed for each of the four human enzymes as defined by gel-filtration column chromatography and by SDS-PAGE. Each of the 37 kDa proteins was identified as a uracil DNA glycosylase by electroelution from the SDS polyacrylamide gel, determination of glycosylase activity by in vitro biochemical assay and identification of the reaction product as free uracil by co-chromatography with authentic uracil. Bloom's syndrome enzymes differed substantially in their isoelectric point and were thermolabile as compared to the normal human enzymes. Bloom's syndrome enzymes displayed a different Km, Vmax and were strikingly insensitive to 5-fluorouracil and 5-bromouracil, pyrimidine analogues which drastically decreased the activity of the normal human enzymes. In particular, each Bloom's syndrome enzyme required 10-100-fold higher concentrations of each analogue to achieve comparable inhibition of enzyme activity. Potential mechanisms are considered through which an altered uracil DNA glycosylase characterizing this cancer-prone human genetic disorder may arise.

A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase.

We have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein (Mr 66,000) that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a beta-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated Mr 36,050 and pI 8.7, corresponding to the Mr 37,000 and pI 8.1 of purified human placental UDG. No homology was seen between this cDNA and the UDG of herpes simplex virus, Escherichia coli, and yeast; nor was there homology with the putative human mitochondrial UDG cDNA or with a second human nuclear UDG cDNA. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated from it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.

Immunological alteration of the Bloom's syndrome uracil DNA glycosylase in Epstein-Barr virus-transformed human lymphoblastoid cells.

The immunological reactivity of the uracil DNA glycosylase was investigated in three Epstein-Barr virus-transformed human lymphoblastoid cell lines. Two were derived from normal human lymphocytes while the third was derived from a Bloom's syndrome patient. A panel of 3 anti-human placental uracil DNA glycosylase monoclonal antibodies (37.04.12, 40.10.09 and 42.08.07) was used. Immunological reactivity was determined in a double-blind enzyme-linked immunosorbent assay (ELISA); by inhibition of enzyme activity; and by immunoblot analysis. In the ELISA, the glycosylase from each lymphoblastoid cell line was recognized by glycosylase antibodies 37.04.12 and 42.08.07. In contrast, antibody 40.10.09 failed to recognize the glycosylase from the Bloom's syndrome cell line. Further analysis demonstrated that the 40.10.09 antibody was unable to inhibit catalysis by the Bloom's syndrome lymphoblast glycosylase. In contrast, the 40.10.09 antibody inhibited the activity of the two normal human lymphoblast enzymes. Denaturation of the Bloom's syndrome lymphoblast glycosylase rendered that protein immunoreactive with the 40.10.09 antibody. These results demonstrated that: (1) the immunological alteration in the Bloom's syndrome uracil DNA glycosylase was detected in hematopoietic cells; and (2) viral transformation did not affect the immunoreactivity of the enzyme from either normal human or Bloom's syndrome cells.

DNA repair and the molecular mechanisms of Bloom's syndrome.

This critical review considers recent work on alterations in DNA repair capacity in Bloom's syndrome as a molecular mechanism for this human disorder. Four main types of DNA repair deficiencies are discussed. These include perturbations in the temporal regulation of DNA repair pathways during the cell cycle, failure to enhance DNA repair pathways during cell proliferation, reduced levels of DNA ligase in Bloom's syndrome cells, and the identification of mutant repair enzyme proteins. These deficiencies are considered in relation to the cellular characteristics of Bloom's syndrome, including delays in DNA replication, hypermutability, and increased incidence of chromosomal aberrations (spontaneously occurring or observed after exposure to environmental agents). The relationship between DNA repair deficiencies and the genetic basis of Bloom's syndrome is described. Previous evidence suggested an autosomal recessive mode of inheritance for Bloom's syndrome. A discussion is presented as to the molecular mechanism through which an alteration in a single gene could result in multiple DNA repair defects.

Hematologic evaluation of employees with leukopenia. Naval Weapons Center, China Lake, California.

Evaluation of 86 employees with a history of leukopenia at the Naval Weapons Center (NWC), China Lake, California, was done by exposure questionnaires, medical histories, physical examinations, peripheral blood smear, and bone marrow evaluations, including morphologic examination, stem cell culture, and cytogenetics. Forty-eight subjects were found to be leukopenic at the time of the study, and two subjects were found to have hairy cell leukemia. All subjects had positive exposure histories and were healthy at the time of the study. Review of peripheral smears identified the patients with marrow abnormalities. Bone marrow cultures revealed several patients with possible marrow suppression. Chromosome studies were not diagnostic. Five-year follow-up health questionnaires revealed no significant health problems; the two workers with hairy cell leukemia are alive and fully functional. Leukopenia in itself does not appear to be a risk factor for poor health, and it is unknown whether or not it may be a useful screening tool to identify workers at risk in toxic environments. Careful evaluation of blood cell counts and peripheral smears should be sufficient to identify people with potential marrow abnormalities.

Immunological lesions in human uracil DNA glycosylase: association with Bloom syndrome.

Three monoclonal antibodies that react with uracil DNA glycosylase of normal human placenta were tested to determine whether one of the antibodies could be used as a negative marker for Bloom syndrome. As defined by enzyme-linked immunosorbent assay, monoclonal antibody 40.10.09, which reacts with normal human glycosylase, neither recognized nor inhibited native uracil DNA glycosylase from any of five separate Bloom syndrome cell strains. Immunoblot analyses demonstrated that the denatured glycosylase protein from all five Bloom syndrome cell strains was immunoreactive with the 40.10.09 antibody. Further, each native enzyme was immunoreactive with two other anti-human placental uracil DNA glycosylase monoclonal antibodies. In contrast, ELISA reactivity was observed with all three monoclonal antibodies in reactions of glycosylases from 5 normal human cell types and 13 abnormal human cell strains. These results experimentally verify the specificity of the aberrant reactivity of the Bloom syndrome uracil DNA glycosylase. The possibility arises that determination of the lack of immunoreactivity with antibody 40.10.09 may have value in the early diagnosis of Bloom syndrome.

Monoclonal antibodies detect conformational abnormality of uracil DNA glycosylase in Bloom's syndrome cells.

The immunoreactivity of normal human and Bloom's syndrome uracil DNA glycosylase was examined using a series of three anti-human placental uracil DNA glycosylase monoclonal antibodies. Immunoreactivity was determined by three separate and independent criteria: enzyme-linked immunosorbent assay (ELISA), enzyme inhibition studies and immunoblot analysis. As defined by each criteria, normal human uracil DNA glycosylase was immunoreactive with each antibody (37.04.12, 40.10.09 and 42.08.07). In contrast, each glycosylase purified from two separate non-transformed Bloom's syndrome cell strains was not reactive with antibody 40.10.09. First, no ELISA reactivity was observed with each glycosylase protein. Second, catalysis by each Bloom's syndrome glycosylase was not inhibited by antibody 40.10.09. However, each Bloom's syndrome enzyme was immunoreactive with antibodies 37.04.12 and 42.08.07. No immunoreactive glycosylase species was observed during the induction of the Bloom's syndrome enzyme during cell proliferation. However, immunoreactivity of the denatured Bloom's syndrome enzyme with 40.10.09 antibody was observed by immunoblot analysis. These results suggest that Bloom's syndrome uracil DNA glycosylase is characterized by a structural alteration in the native glycosylase protein secondary to the primary antigenic site recognized by the 40.10.09 antibody. This altered antigenicity may provide an immunological marker for the identification of this human genetic syndrome.

Purification and properties of the human placental uracil DNA glycosylase.

Human placental uracil DNA glycosylase was purified 3700-fold to apparent homogeneity as defined by SDS gel analysis. Its immunological characteristics were examined using three monoclonal antibodies prepared against partially purified human placental uracil DNA glycosylase. Immunoblot analysis demonstrated that, even in crude isolates, only one glycosylase species of molecular weight 37,000 could be detected. Each of the three monoclonal antibodies quantitatively recognized the highly purified enzyme by ELISA. The glycosylase is a single polypeptide with a molecular weight of 37,000 as defined by both Sephadex gel filtration and by SDS-polyacrylamide gel electrophoresis analysis. The enzyme is heat-stable, with a t 1/2 of greater than 30 min at 42 degrees C or at 45 degrees C. Surprisingly, inhibitor analysis demonstrated that the glycosylase was inhibited by preincubation with either 5-fluorouracil or 5-bromouracil. However, no significant inhibition was observed when either compound was added directly to the enzyme assay.

Physical association of the human base-excision repair enzyme uracil DNA glycosylase with the 70,000-dalton catalytic subunit of DNA polymerase alpha.

A monoclonal antibody prepared against a partially purified human uracil DNA glycosylase was found, on further purification of the enzyme, to be inactive against the glycosylase. However, immunoreactivity was observed in other protein fractions that contained DNA polymerase activity. The immunoreactive protein was purified to homogeneity and identified as a catalytic subunit of DNA polymerase alpha by molecular mass, by aphidicolin sensitivity, and by recognition by a monoclonal antibody against human KB cell DNA polymerase alpha. Our monoclonal antibody had no effect on homogeneous human uracil DNA glycosylase activity but severely inhibited the activity of the homogeneous human DNA polymerase alpha catalytic subunit. The suspicion that the two proteins were physically associated was confirmed by finding that, on mixing the DNA polymerase alpha subunit with the glycosylase, the latter was strongly inhibited by our monoclonal antibody. These results demonstrate that this monoclonal antibody recognizes not only the DNA polymerase alpha subunit but also the uracil DNA glycosylase when it is physically attached to the polymerase subunit. These results contribute to the definition of relationships between those proteins that may comprise the human base-excision repair multienzyme complex.

Testicular tumor in patient with persistent müllerian duct syndrome.

A case of a yolk-sac tumor associated with an intra-abdominal testis in a patient with persistent müllerian duct syndrome is presented.

A, B and H antigens in normal urothelium: an immunohistochemical study using monoclonal antibodies with the avidin-biotin complex technique.

Immunohistochemical staining for the A, B and H blood group antigens was studied in 61 normal human ureters using monoclonal antibodies with avidin-biotin complex application. Thirty-seven of these were archival material, and 24 were processed prospectively. In 100 per cent of the prospectively processed ureters, A, B and H antigens were demonstrated corresponding to the blood type of the source. Archival material stained for A, B and H 65 per cent, 50 per cent and 100 per cent of the time, respectively. Serial sampling of prospectively processed ureters showed diminution of staining with prolongation of immersion in formalin. A characteristic staining pattern was found in ureters from patients with type B blood.

A comparison of total white blood cell counts on the Technicon H6000 and Coulter Counter Model ZBI in an occupational health program.

White blood cell (WBC) counts obtained using a Technicon H6000 in a large occupational health program were compared with counts obtained on identical samples using a Coulter Counter Model ZBI. To assure quality control and to assess the comparability of results obtained on the two machines, a 25% systematic sample (N = 586) of analyses performed on the Technicon were performed independently and blindly on the Coulter Counter. The Pearson product-moment-correlation between WBC counts from the two machines was r = 0.94 (P less than or equal to 0.0001). The mean WBC count was 6,664 cells/mm3 for the Technicon and 6,790 cells/mm3 for the Coulter Counter. The estimated prevalence of leukopenia (WBC counts less than or equal to 4,500 cells/mm3) was 10.6% using the Technicon and 9.7% using the Coulter Counter. The results demonstrate that the two machines provide results that can be compared directly without corrections.

Experimental transmission of duck hepatitis B virus.

Susceptibility to experimental infection with duck hepatitis B virus (DHBV) was explored, with the objective of defining procedures that were both rapid and reproducible. For the purpose of these experiments, a small flock of DHBV-free breeders was established as a source of susceptible eggs and ducklings, since ca. 10% of the ducks (all ages) from commercial flocks were DHBV infected. Intravenous inoculation of DHBV into 15-day duck embryos from the DHBV-free flock produced a persistent infection, with a high-titer viremia, in at least 80% of the injected animals. The tissue tropism of DHBV in these experimentally infected animals was similar to that associated with natural, congenital infections from viremic ducks to their progeny. Virus antigen was found not only in hepatocytes and bile duct epithelium of liver, but also in cells associated with exocrine and endocrine pancreas, and in proximal convoluted tubular epithelium of kidney. Infection of embryonic liver was rapid, as evidenced by active synthesis of DHBV-DNA by reverse-transcription of RNA by 24 hr postinjection. During this latter analysis, formation of supercoiled viral DNA appeared to precede the reverse-transcription phase of viral DNA synthesis, suggesting that this species may be important in initiation of infection.