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Biomedical subjects

J G Gray

Publications and source records attributed to J G Gray.

At least 19 recordsLinked to original sources

Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.

Centrifugation, Density Gradient

Three-dimensional imaging for brachytherapy planning.

A computerized 3-D brachytherapy treatment planning program has been developed which simulates catheter trajectories, radioisotope seed positioning and percentage dosimetry zones about a catheter/seed array in relationship to CT or MRI images. The high activity 125I seed is used in this program. A visual and a non-linear mathematical optimization program will also be described.

Brachytherapy

Magnetic resonance imaging and anatomic atlas mapping for thalamotomy.

A computerized anatomic atlas mapping technique has been developed and used to determine target sites for stereotactic thalamotomies. The digitized stereotactic atlases of Schaltenbrand and Bailey, Van Buren and Borke, and Schaltenbrand and Wahren are used in conjunction with an associated electrophysiological data base to determine the intended target site on MR images of the diencephalon. This mapping technique uses the 'Brierley and Beck' proportional hypothesis for thalamic nuclear determination. Target site determination by this method was found to be very accurate, and frequently the mere placement of the probe in the target site resulted in tremor cessation.

Brain Mapping

Three-dimensional whole-brain mapping.

The implementation and use of a computerized whole-brain mapping system which can be used in conjunction with MRI, CT, angiographic and other brain imaging techniques is described. Three mapping systems based upon common reference structures about the third and fourth ventricular core of the brain are used in conjunction with internationally recognized nomenclature to create a normalized whole-brain mapping system according to the Talairach/Tournoux proportional grid technique.

Brain

Human immunodeficiency virus infection and syncytium formation in HeLa cells expressing glycophospholipid-anchored CD4.

The CD4 molecule, a glycoprotein expressed primarily on the cell surface of specific T lymphocytes, is thought to function in T-cell antigen recognition and activation. In addition, CD4 serves as a receptor for human immunodeficiency virus type 1 (HIV-1) by a direct interaction with the HIV-1 surface glycoprotein (gp120). To further characterize the HIV-1-cell interaction, a HeLa cell line was established that expressed a chimeric molecule of CD4 and decay-accelerating factor (DAF). In the chimeric CD4-DAF molecule the transmembrane and cytoplasmic domains of CD4 were deleted and replaced with the carboxy-terminal 37 amino acids of DAF. This resulted in the anchoring of the extracellular domain of CD4 to the cell membrane via a glycophospholipid linkage. The glycophospholipid-anchored CD4 had a molecular size of approximately 56 to 62 kDa and was released following treatment of the cells with phosphatidylinositol-specific phospholipase C. HeLa cells expressing the CD4-DAF hybrid could be infected with HIV-1, as evidenced by reverse transcriptase activity, p24 core antigen content, and infectious virus production. In addition, transfection of the HeLa CD4-DAF cells with a plasmid that directs the synthesis of HIV-1 envelope glycoproteins or cocultivation with HeLa cells expressing the virus glycoproteins resulted in syncytium formation. These results indicate that the transmembrane and cytoplasmic domains of the CD4 molecule are dispensable for both HIV infection and syncytium formation.

Acquired Immunodeficiency Syndrome

A new breast screening programme--an audit of the first year.

The experience of a new breast screening unit is reported. In the first 12 months there was a 72.1% response rate with 9890 women being screened. After the initial screen 598 (5.8%) were referred to the Assessment clinic. Following repeated mammography and clinical examination 166 patients were referred for a surgical opinion; 105 (1.06% of the total screened) were considered to be possibly malignant; 61 further patients with clinical and mammographically benign disease were kept under review by a single surgeon; 101 patients had excision biopsies; 28 of 32 palpable breast lumps and 36 of 69 non-palpable lesions proved to be malignant.

Biopsy

Stomal adenocarcinoma in Crohn's disease.

Malignant change occurring at the site of a stoma in two patients with proved Crohn's disease is described. Patients with ulcerative colitis have an increased risk of colonic malignancy and Crohn's disease is also associated with both small and large bowel carcinoma. Most previous reports of stomal carcinoma have been associated with ulcerative colitis although Crohn's disease seems to carry a greater risk of associated small bowel carcinomas. This is the first report of stomal carcinoma complicating Crohn's disease. Epithelial dysplasia is associated with gastrointestinal carcinomas in both ulcerative colitis and Crohn's disease and a dysplasia-carcinoma sequence has been suggested as the origin of these tumours. In both our patients with stomal adenocarcinoma, dysplasia was identified in adjacent tissues, which suggests a similar mechanism. Malignant change should be suspected if epithelial dysplasia is discovered in a biopsy specimen from the mucosa of an ileostomy in Crohn's disease, and this risk is increased if the dysplasia is of a high grade.

Adenocarcinoma

Aspiration cytology and ultrasonography of cold thyroid nodules.

Over an 18 month period, 50 'high risk' patients with solitary or dominant cold thyroid nodules on 99m Tc-pertechnetate scanning, have undergone fine needle aspiration biopsy cytology (ABC) under general anaesthetic prior to thyroidectomy. Histological malignancy was confirmed in only four patients (8%). Cytological malignancy was suspected or confirmed in six patients, each having a false positive rate of 4%. There were no false negative reports. Ultrasonography, performed pre-operatively in 37 of the patients, did not significantly add to the overall patient management. ABC appears to be safe, simple and sufficiently accurate to incorporate its use routinely in the pre-operative assessment of thyroid nodules.

Adult

An Alcaligenes faecalis isolate from turkeys: pathogenicity in selected avian and mammalian species.

An Alcaligenes faecalis isolate of known pathogenicity for turkeys was examined for adherence and cytotoxicity in tracheal organ cultures of turkeys, chickens, Japanese quail, guinea pigs, hamsters, and mice, and for colonization and pathogenicity in these 6 species. Adherence and colonization were detected by fluorescent antibody staining. Infected and noninfected tracheal rings were examined by phase-contrast microscopy for cytotoxicity (ciliostasis, blebing of the cell membrane, and sloughing of the ciliated epithelium). Alcaligenes faecalis adhered to the tracheal rings of all species examined. Cytotoxicity was apparent in the tracheal rings of turkeys, quail, and chickens. Cytotoxicity was not detected in tracheal rings from the mammalian species. Alcaligenes faecalis colonization of turbinates and tracheas of intact turkeys and quail was detected. Clinical signs of alcaligenes rhinotracheitis were observed and histopathologic characteristics of the disease were detected. Chickens, guinea pigs, hamsters, and mice were refractory to infection with this isolate of A faecalis.

Adhesiveness

Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.

The initiation of DNA replication of plasmid R6K is triggered by a 35-kilodalton initiator protein. The initiator protein had been elusive because of its lability and the lack of a convenient assay procedure to aid its purification. Using recombinant DNA techniques, we have fused the cistron of the initiator near its COOH-terminal end, in the correct reading frame, to the lacZ cistron of Escherichia coli at the ninth codon from the NH2 terminus. The fused cistron yielded a protein that was not only stable in vivo but also had dual activities: initiation of DNA replication in vivo and in vitro and hydrolysis of beta-galactoside. Using an affinity column that is specific for beta-galactosidase, we have demonstrated the rapid purification of the hybrid protein to near homogeneity. Exploiting the polymeric structure of the initiator, we have also isolated the nonfused form of the initiator protein, associated through subunit interaction with the beta-galactosidase-fused protein, which permits its purification by affinity chromatography. NH2-terminal amino acid sequence analysis of the heteropolymer has not only shown that the fused and nonfused initiators have the same sequence but also confirmed the protein sequence of the initiator as predicted from its nucleotide sequence. The techniques described here should be generally useful for the isolation of other proteins that are difficult to purify by conventional procedures.

Bacterial Proteins

Pathogenesis of change in the upper respiratory tracts of turkeys experimentally infected with an Alcaligenes faecalis isolate.

The course of changes within the upper respiratory tracts of turkey poults experimentally infected with Alcaligenes faecalis was studied. The initial change observed (5 days post-inoculation) was colonization of the upper respiratory tract by the bacterium. Changes in the nasal turbinates and trachea were first apparent as a focal loss of cilia but subsequently developed into a general loss of cilia (11 days post-inoculation). Eventually, the entire ciliated epithelial layer in the cranial region of the trachea was lost (13 days post-inoculation). With the loss of cilia and ciliated cells, a highly viscous mucus was able to accumulate in the anterior one-half to two-thirds of the trachea. In addition, changes in the gross structure of the trachea (flaccid trachea) were observed in all poults inoculated with A. faecalis. There was an apparent gradation in the severity of these changes from severe in the cranial region of the trachea to mild in the region just anterior to the bronchial bifurcation. The observations resulting from A. faecalis infection indicated two major tracheal changes responsible for the chronic and sometimes severe nature of this disease. These changes included a loss of ciliary activity and a flaccid trachea which together resulted in the accumulation and stasis of mucus and tracheal collapse.

Alcaligenes

The effect of topical povidone iodine on wound infection following abdominal surgery.

The effect of povidone iodine on wound sepsis following gastrointestinal surgery was studied in a trial involving 153 patients of whom 72 had their wounds sprayed with povidone iodine dry powder (Disadine DP) and 81 acted as a control group. The infection rate of 9.9 per cent in the patients treated with povidone iodine was significantly lower than that of 24.4 per cent in the control group (P less than 0.05). Bacterial contamination of the wound at the time of operation was shown to be of importance, being associated with a 52 per cent infection rate in the control group. However, spraying of contaminated wounds with povidone iodine reduced the infection rate to the significantly lower level of 11 per cent (P less than 0.05). We conclude that povidone iodine is a safe and effective means of reducing wound sepsis following gastrointestinal surgery.

Administration, Topical

Value of 1-alpha-hydroxy vitamin D3 in treatment of primary hyperparathyroidism before parathyroidectomy.

The postoperative course of six patients with primary hyperparathyroidism and obvious radiological evidence of bone disease pretreated with 1-alpha-hydroxy vitamin D3 (1 alpha HCC) was indistinguishable from that of six patients with a similar clinical and radiological picture who were not pretreated. 1alphaHCC may increase the hypercalcaemia in some cases and cannot be recommended for the routine preparation of such patients for surgery.

Adolescent