DNA mutagenesis and recombination.
The polymerase chain reaction is used for site-specific mutagenesis and for DNA recombination without any enzymatic reaction in vitro, apart from DNA amplification.
DNA, Circular↗
Biomedical subjects
D H Jones
Publications and source records attributed to D H Jones.
Trauma services requirements in a district general hospital serving a rural area.
OBJECTIVE: To assess the demands made on a regional trauma centre by a district trauma unit. DESIGN: Two part study. (1) Prospective analysis of one month's workload. (2) Retrospective analysis of one year's workload by using a computer based records system. Comparison of two sets of results. SETTING: Accident unit in Gwynedd Hospital, Bangor. PATIENTS: (1) All patients who attended the accident unit in August 1988. (2) All patients who attended the accident unit in the calendar year April 1988-April 1989. MAIN OUTCOME MEASURE: Workload of a district trauma unit. RESULTS: In August 1988 there were 2325 attendances; 2302 of these were analysed. In all, 1904 attendances were for trauma; 213 patients were admitted to the trauma ward and 103 required an operation that entailed incision. Patients who attended the unit had a mean (range) injury severity score of 2-13 (0-25). Only two patients had injuries that a district general hospital would not be expected to cope with (injury severity score greater than 20). In the year April 1988-April 1989, 21,007 patients attended the unit. In all, 17,958 attendances were for orthopaedic injuries or injuries caused by an accident; 1966 patients were admitted to the unit. CONCLUSIONS: Most trauma is musculoskeletal and relatively minor according to the injury severity score. All but a few injuries can be managed in district general hospitals. In their recent report the Royal College of Surgeons has overestimated the requirements that a British district general hospital would have of a regional trauma centre.
Catchment Area, Health↗
Subcellular distribution and mobilization of MAC-1 (CD11b/CD18) in neonatal neutrophils.
The CD11b/CD18 (Mac-1) heterodimeric surface glycoprotein contributes to a broad range of adherence-dependent neutrophil inflammatory functions. Previous investigations have indicated that diminished expression or regulation of Mac-1 may underlie abnormalities of stimulated adhesion and chemotaxis of neonatal neutrophils in vitro and inflammatory deficits in human neonates. To define the pathogenic mechanisms contributing to these findings, we compared the distribution and translocation of Mac-1 in subcellular fractions of neonatal and adult neutrophils before and after chemotactic stimulation. The total cell content of Mac-1 and the proportions of Mac-1 in beta fractions (vitamin B12 binding protein-rich granules), pre-gamma fractions (gelatinase-rich granules), or gamma fractions (plasma membrane) of neonatal neutrophils were comparable with those of adult neutrophils. However, after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10 nmol/L, 37 degrees C, 15 minutes), neonatal neutrophils demonstrated (1) diminished translocation of Mac-1 from pre-gamma fractions (P less than .05), and (2) diminished surface expression of Mac-1 (P less than .05), as compared with healthy adult neutrophils. As shown in enzymatic and immunochemical assays, neonatal cells contained significantly (P less than .01) diminished levels of neutrophil gelatinase. In response to FMLP (0.1 to 10 nmol/L, 37 degrees C, 15 minutes), neonatal suspensions also released significantly (P less than .001) less gelatinase, as compared with adult neutrophil suspensions. These observations demonstrate that diminished mobilization of Mac-1 from gelatinase-rich granular pools in neonatal neutrophils is associated with abnormal surface expression of this glycoprotein after chemotactic stimulation. This abnormality may contribute, in part, to abnormal migratory properties of neonatal neutrophils in response to inflammatory stimuli.
Antigens, Differentiation↗
High dose intensity combination chemotherapy for advanced epithelial ovarian carcinoma: results of a pilot study.
Retrospective studies have recently demonstrated a significant correlation between dose intensity of chemotherapy and response rates and survival in various diseases including epithelial ovarian carcinoma. As part of a proposed randomised trial to assess the effect of dose intensity on outcome in ovarian carcinoma, a pilot study has been undertaken to determine the toxicity and efficacy of the high intensity therapy. Nineteen patients with advanced ovarian carcinoma received initial treatment with cisplatin 120 mg m-2 i.v. day 1, and cyclophosphamide 1,000 mg-2 i.v. day 1, given at 21-day intervals for six cycles. The average relative dose intensity of this therapy is 1.14 when compared with the CHAP regimen. Severe toxicity was experienced by most patients. The median received average relative dose intensity was 0.90, with only one patient receiving treatment to the proposed intensity. Randomised studies of the effect of dose intensity in ovarian carcinoma are essential, but an initial step must be to assess whether the proposed high dose treatment can be delivered.
Adult↗
Health risk factors among the Amish: results of a survey.
The Amish are a unique societal subgroup deliberately setting themselves apart from the modern world through religious beliefs and practices. Few reports and no systematic studies have been conducted on Amish health behaviors. A representative sample of 400 Amish adults residing in Holmes County, Ohio, were interviewed using the Behavioral Risk Factor Survey (BRFS). For comparison, a representative sample of 773 non-Amish adults responded to the same survey through telephone interviews. Prevalence of self-reported hypertension was lower among Amish than non-Amish, obesity was greater among Amish than non-Amish women but similar among men, and stress responses were more prevalent among Amish than non-Amish. While some differences between groups could be influenced by response biases, many are supported by less systematic observations of Old Order Amish societies. Patterns of health behavior reflect characteristics of Amish culture and may be responsible for certain favorable mortality rates among the Amish population. Need for cultural sensitivity is evident in health promotion program design and implementation.
Adolescent↗
A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles.
Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase chain reaction was used to generate products that when combined, denatured and reannealed, form double-stranded DNA with discrete, cohesive single-stranded ends. The generation of these cohesive ends of DNA permits the formation of precise, directional DNA joints without dependence on enzyme restriction sites. The primers were designed such that these cohesive single-stranded ends annealed to form circular DNA. The recombinant of interest was generated following only 14 amplification cycles. These recombinant circles of DNA were directly transfected into E. coli. In the mutagenesis protocol, the desired mutant was obtained at 83%-100% efficiency. Unwanted mutations were not detected, indicating a less than 0.025% nucleotide misincorporation frequency. In the directional subcloning protocol, inserts were positioned precisely in the recipient plasmid and were in the correct orientation. One unwanted mutation was detected after sequencing 900 bases, indicating a 0.11% nucleotide misincorporation frequency. Each manipulation, from setting up for the DNA amplification to transfection into E. coli. can easily be accomplished in one day.
Base Sequence↗
Osteoporosis and immunosuppression in multiple myeloma.
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Aged↗
Monoclonal antibodies as targeting agents for cytotoxic compounds in vivo: a current assessment.
Monoclonal antibodies have proved useful in the in vitro diagnosis of tumour type. Radiolabelled antibodies have also been investigated as targeting agents for the in vivo diagnosis of tumour metastasis. These studies have revealed the potential of using 'targeting agents' to identify tumours, but in addition have exposed some of the problems associated with the technique. This review describes our experiences using radiolabelled antibodies for the identification and treatment of tumours arising from the neuro-ectoderm. Based on a small number of 4 patients studies, we conclude that there are limitations on the use of radiolabelled antibodies for targeting isotopes to primary tumour masses. In addition, we outline a possible important role for antibodies in targeting therapeutic agents to minimal residual disease and diffuse tumours presenting as micro-metastasis.
Animals↗
The role and mechanism of action of 13-cis-retinoic acid in the treatment of severe (nodulocystic) acne.
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Acne Vulgaris↗
ChlVPP chemotherapy in advanced Hodgkin's disease.
Between March 1978 and January 1987 54 patients with advanced Hodgkin's disease (HD) or relapse following radiotherapy (RT) for Hodgkin's disease have been treated with combination chemotherapy consisting of chlorambucil, vinblastine, procarbazine and prednisolone (ChlVPP). A subgroup of five patients with bulky mediastinal disease received mantle RT in addition to ChlVPP chemotherapy. Forty-two patients (77.8%) entered complete remission with 33 (61.0%) remaining in unmaintained remission and 44 (81.5%) alive at a median follow up of 51 months (range: 22-103). The treatment was generally well tolerated with minimal toxicity. ChlVPP is effective first-line treatment for Hodgkin's disease with results which may be comparable to those achieved for MOPP but with significantly less toxicity.
Adult↗
Primary cutaneous neuroendocrine (Merkel cell or trabecular carcinoma) tumour of the skin: a radioresponsive tumour.
Four patients with primary neuroendocrine carcinoma of the skin (Merkel cell or trabecular carcinoma) are reported to document their response to radiotheraphy. In three patients there was complete response following radiation treatment, with no local recurrence, whilst the other patient died later with distant metastases. In one patient, post-operative irradiation after excision of an involved local node prevented the growth of further tumour in this nodal group but the unirradiated primary site recurred after initial wide local excision only. This rare tumour appears to be radioresponsive, and although more clinical data are required, we would advocate a wider study of radiotherapy to the primary lesion after biopsy or excision biopsy, in association with prophylactic nodal irradiation. These tumours occur predominantly in elderly people in whom the avoidance of extensive surgery is particularly desirable.
Aged↗
Involvement of the ileocaecal region by non-Hodgkin's lymphoma in adults: clinical features and results of treatment.
Between January 1977 and January 1988, 19 patients with non-Hodgkin's lymphoma (NHL) involving the ileocaecal region were cared for by the CRC Wessex Medical Oncology Unit. Fifteen of these patients had primary ileocaecal NHL (stages IE or IIE) and four had secondary involvement of this region (stage IV). The commonest clinical presentation was with abdominal pain and a palpable mass in the right iliac fossa. Bulky (greater than 10 cm) disease was a particularly common feature, and complete surgical removal was possible in only seven patients. All patients had intermediate (18) or high grade (one) NHL using the Working Formulation. The commonest histological subtype was diffuse large cell. Seventeen patients received postoperative therapy, comprising local radiotherapy in one and combination chemotherapy in the remaining 16. Eleven of the 19 patients remain disease-free 6-60 months from diagnosis. Because of the high incidence of bulky disease at this site, postoperative therapy may be indicated, even for patients with apparently completely excised stage I disease.
Adult↗
The activity of phosphoenolpyruvate carboxykinase throughout the lactation cycle of the guinea pig mammary gland.
The enzyme phosphoenolpyruvate carboxykinase (PEPCK) has been measured in the guinea pig mammary gland throughout the pregnancy-lactation cycle. This is of interest since the primary importance of PEPCK is thought to be its role in gluconeogenesis and it is questionable whether or not gluconeogenesis occurs in the mammary gland. The enzyme activity, present in both the cytosol and mitochondria, was shown to follow the lactation profile. During the transition into lactation, cytosolic PEPCK activity increases 11-fold and mitochondrial PEPCK activity 43-fold while tissue weight increases 4-fold. Fructose 1,6-bisphosphatase was found to increase at a rate only slightly greater than that of the tissue weight. The increase in mitochondrial PEPCK activity is thus about 10 times greater than that of general tissue expansion, whereas the cytosolic PEPCK activity increase is only 2-fold greater. The activity of fructose 1,6-bisphosphatase appears to be merely keeping pace with general tissue expansion. The mitochondrial enzyme constitutes 59 +/- 3% of the total gland PEPCK activity in the prepartum state and 86 +/- 2% at midlactation. Therefore, mitochondrial PEPCK is the isoenzyme undergoing the greater increase during the transition into lactation in the guinea pig mammary gland and thus would appear to play the more important role in the conversion of oxalacetate to phosphoenolpyruvate in this tissue.
Animals↗
Disability glare and hydrogel lens wear--revisited.
Decreased tolerance to glare is a common complaint among hydrogel contact lens wearers. Recently this subjective complaint was measured and quantified in our laboratory in five subjects by determining increment thresholds as a function of background luminance and glare source location for both spectacles and hydrogel lens refractive corrections. Additional testing, using the same apparatus and similar protocols, has not entirely supported the original result. After testing a total of 15 subjects, data averaged across all subjects revealed no significant hydrogel contact lens-induced increases in disability glare; however, analysis of individual data revealed three classes of effects within the population tested. Subjects either demonstrated a significant glare effect induced by contact lens wear (6 of 15), no increase in disability glare (7 of 15), or a significant decrease in disability glare (2 of 15).
Adult↗
Quantitation of intracellular Mac-1 (CD11b/CD18) pools in human neutrophils.
The adhesive glycoprotein Mac-1 (CD11b/CD18) of the CD11/CD18 complex contributes to multiple neutrophil inflammatory functions. Activation of neutrophils by chemotactic stimuli results in a rapid, protein synthesis-independent increase in surface Mac-1 derived from incompletely defined intracellular compartments. Therefore, we developed a novel quantitative lectin immunoblot technique to define intracellular pools of Mac-1 in subcellular neutrophil fractions resolved on discontinuous Percoll gradients. In cavitates of unstimulated neutrophils, 30% and 26% of total Mac-1 was identified in beta [1.10 gm/ml; vitamin B12 binding protein (vit B12 B.P.)-rich] or pre-gamma (1.07 gm/ml; vit B12 B.P.-poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane-rich gamma (1.06 gm/ml) fractions. N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation (10(-8) M, 15 min, 37 degrees C) significantly diminished Mac-1 in pre-gamma (-18% of total, P less than 0.05) but not beta fractions (+6% of total). Under these conditions, the content of Mac-1 in gamma fractions increased 13% in association with four- to eightfold increase in surface Mac-1 expression (OKM-1 binding). These findings suggest that chemotactic stimuli increase plasma membrane and/or surface Mac-1 on human neutrophils by mobilizing a novel intracellular granule pool.
Adult↗
Two routes for 99mTc-DMSA uptake into the renal cortical tubular cell.
Critical factors determining the renal handling of 99mTc-dimercaptosuccinic acid (DMSA) are protein binding in plasma and the renal 99mTc-DMSA extraction efficiency. Comparison of the count rate over soft tissue with that over the cardiac blood pool about 1 h after injection demonstrated that 99mTc-DMSA is not exclusively an intravascular label. 99mTc-DMSA was 76% protein bound in plasma as demonstrated by HPLC and gel filtration. Assuming that the 24% that is not protein bound is filtered at the glomerulus, the renal extraction efficiency of 99mTc-DMSA by glomerular filtration is about 5%. Since the total renal extraction efficiency was also found to be about 5%, the majority of the activity that becomes fixed in the renal cortex arrives there as a result of filtration followed by tubular reabsorption rather than by direct extraction from peritubular blood. However, discordant changes in DMSA and DTPA uptake induced by captopril in renovascular hypertension (RVH) suggested that a minority of uptake was by direct peritubular extraction. This kinetic model was supported by indirect measurement of protein binding and extraction efficiency based on the kinetics of 99mTc-DMSA disappearance from plasma and kinetics of uptake in the kidneys. Furthermore, differential functional studies based on 99mTc-DMSA and 99mTc-DTPA before and after captopril in patients with RVH due to unilateral renal artery stenosis confirmed filtration followed by tubular reabsorption as the predominant route for DMSA uptake by the kidney.
Adult↗
An unusual weightlifting injury.
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Adult↗
Mutagenic potential of binary mixtures of nitro-polychlorinated dibenzo-p-dioxins and related compounds.
The mutagenic potential of binary mixtures of nitro-polychlorinated dibenzo-p-dioxins and other environmentally related compounds was determined using Salmonella typhimurium strain TA98 in the standard plate incorporation assay. Samples tested included binary mixtures of 4-nitro-4'-chlorobiphenyl with 6-nitro-4,2',3',4',5'-pentachlorobiphenyl, 4-nitrobenzo-p-dioxin with 4-nitro-2,3,8-trichlorodibenzo-p-dioxin, and benzo[a]pyrene with either nitropentachlorobiphenyl or nitrotrichlorodibenzo-p-dioxin. Inhibition was the primary interaction observed for the mixtures of polyhalogenated dioxins or biphenyls with the direct-acting mutagens nitrodibenzo-p-dioxin or nitrochlorobiphenyl. At the highest dose tested, nitrotrichlorodibenzo-p-dioxin inhibited the bacterial mutagenicity of nitrodibenzo-p-dioxin by almost 50%, while pentachlorobiphenyl inhibited the mutagenicity of nitrobiphenyl by 34%. Conversely, synergism was the primary interaction observed for mixtures of halogenated aromatics with the promutagen benzo[a]pyrene. The addition of nitrotrichlorodioxin to benzo[a]pyrene enhanced the mutagenicity of the latter compound by as much as 70%, while the mutagenic potential of a mixture of benzo[a]pyrene and nitropentachlorobiphenyl was approximately 50% greater than the mutagenicity of benzo[a]pyrene alone. In summary, mixtures of nonmutagenic nitropolyhalogenated biphenyls or dibenzo-p-dioxins appear to inhibit the mutagenicity of direct-acting mutagens, while these same compounds seem to enhance the mutagenic potential of the promutagen benzo[a]pyrene.
Animals↗