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Biomedical subjects

M Ward

Publications and source records attributed to M Ward.

At least 37 records · Page 2Linked to original sources
PubMed1991

A comparison of patient rewarming devices after cardiac surgery.

Three regimens for rewarming patients after cardiac surgery involving hypothermic cardiopulmonary bypass were studied in 30 patients. The control group (n = 10) received no active rewarming, the oesophageal group (n = 10) was warmed centrally using an oesophageal heat exchanger and the radiant group (n = 10) was warmed peripherally with an overhead radiant heater. There were no statistically significant differences between the groups apart from the higher mean skin temperatures in the peripheral group.

Body Temperature↗
PubMed1991

Somatic gene therapy.

Many problems obviously continue to exist in gene transfer using retroviruses as a means of inserting foreign DNA into hematopoietic stem cells, especially with regulated genes such as the human beta globin genes. First, it is unclear whether the available retroviral vectors will infect enough stem cells for gene transfer to be successful over the long term. Second, there may be sequences necessary for normal beta globin gene expression that may also inhibit the normal retroviral life cycle, thus decreasing the efficiency of gene transfer or gene expression. It seems clear that in order to optimize the success of gene transfer, the highest possible titer of viral production is necessary. New approaches are aimed at increasing viral titer. The transfect/infect method appears useful. Growth factors may also be useful by increasing stem cell proliferation. Single growth factors may not be sufficient to optimize stem cell cycling. To date, interleukin-3 seems to be the single most useful growth factor, although interleukin-3 with interleukin-6 or other combinations of growth factors including interleukin-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) appear to have potential. Future work also is required to optimize the number of marrow stem cells needed for successful transplantation. Long-term bone marrow culture and stromal cell cultures may provide new and improved marrow culture conditions for achieving this goal. Improvements in the efficiency of both gene transfer and gene expression are necessary before we can consider the concept of gene transfer for the treatment of various hematologic genetic diseases in humans.

Animals↗
PubMed1990

Granulocyte-macrophage colony stimulating factor from human lymphocytes. The effect of glycosylation on receptor binding and biological activity.

Native human granulocyte-macrophage colony stimulating factor (hGM-CSF) has previously been purified using methods which typically required several sequential chromatographic steps and only yielded small amounts of hGM-CSF. We have purified and characterized hGM-CSF using monoclonal antibodies raised against bacterially synthesized hGM-CSF. Activated donor T-lymphocytes grown in interleukin-2 and then reactivated with phytohemagglutinin produce several forms of hGM-CSF which can be purified using immunoaffinity absorption followed by reversed phase high performance liquid chromatography. The purified hGM-CSF consisted of at least nine species ranging in molecular weight (Mr) from 14,500 to 32,000. The higher Mr forms contained one or two N-linked carbohydrate moieties and were more acidic by two-dimensional Western blot analysis, consistent with increasing sialation. N-terminal sequence analysis of high and low molecular weight hGM-CSF fractions corresponded to that predicted by the cDNA sequence. Using the AML 193 [3H]thymidine incorporation assay the specific activity of the heavily glycosylated hGM-CSF was 1 x 10(8) units/mg compared with 6 x 10(8) units/mg for the non-glycosylated hGM-CSF produced by Escherichia coli. The different hGM-CSF forms induced neutrophil superoxide anion production by a variable amount depending on the extent of N-linked glycosylation. Receptor binding studies demonstrated lower receptor affinity for the heavily glycosylated form (KD = 820 pM) compared to less heavily glycosylated (KD = 78 pM) and non-glycosylated hGM-CSF produced by E. coli (KD = 30 pM). These differences are due to differences in the kinetic association rate.

Binding, Competitive↗
PubMed1990

Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori.

We have cloned genomic pepA sequences encoding the aspartic proteinase aspergillopepsin A (PEPA) from Aspergillus awamori using a synthetic oligodeoxyribonucleotide probe. Nucleotide sequence data from the pepA gene revealed that it is composed of four exons of 320, 278, 249, and 338 bp. Three introns which interrupt the coding sequence are 51, 52, and 59 bp in length. Directly downstream from the putative start codon lies a sequence encoding 69 amino acids (aa) which are not present in mature PEPA. Based on similarities to other aspartic proteinases, this region may represent a 20-aa signal peptide followed by a 49-aa propeptide that is rich in basic aa residues. Northern blots of total cellular RNA extracted from A. awamori cells indicate that pepA is transcribed as a single 1.4-kb mRNA. Mutants of A. awamori lacking the pepA structural gene were derived by the following gene replacement strategy. First, we constructed a plasmid in which a 2.4-kb SalI fragment containing the entire pepA coding region was deleted from a 9-kb Eco RI genomic DNA clone and replaced by a synthetic DNA polylinker. Second, a selectable argB gene was inserted into the polylinker. Third, the EcoRI fragment which contained the argB marker flanked by pepA sequences was excised from the plasmid and used to transform an argB auxotroph of A. awamori. From 16-40% of the resulting prototrophic transformants were found to have a PEPA-deficient phenotype when screened with an immunoassay using antibodies specific for PEPA. Southern hybridization experiments confirmed that these mutants resulted from a gene replacement event at the pepA locus.

Amino Acid Sequence↗
PubMed1990

Percutaneous endoscopic gastrostomy: a two-year experience.

The results of percutaneous endoscopic gastrostomy in 25 patients who required enteral alimentation when feeding by mouth was not possible is reported. The procedure was successful on 30 occasions, with a low incidence of major complications. Endoscopic gastrostomy is a simple, less expensive alternative to surgical gastrostomy with a lower morbidity and mortality.

Adolescent↗
PubMed1990

Cloning, characterization, and expression of two alpha-amylase genes from Aspergillus niger var. awamori.

Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.

Amino Acid Sequence↗
PubMed1990

Regulation of the glaA gene of Aspergillus niger.

The glucoamylase gene of Aspergillus niger, glaA, is expressed at high levels in the presence of starch. We have determined the nucleotide sequence of 1966 bp of the 5' flanking region of the glaA gene and have begun to identify sequences important for the control of glaA expression by deletion analysis. Constructs containing deletions extending into the glaA gene promoter were introduced into an A. niger host whose own glaA gene had been disrupted by a gene replacement event. Secreted levels of glucoamylase, expressed from each of the recombinant glaA genes, were measured by enzyme immunoassay. The effect of each deletion on the expression of glaA, when grown on differing carbon sources, was used to determine the limits of sequences upstream of glaA responsible for gene regulation. A region between -562 and -318 appears to direct high-level expression, whereas only 214 bp of 5' flanking sequence is required to initiate the start to transcription.

Amino Acid Sequence↗
PubMed1990

Rectal bleeding. Patient delay in presentation.

Patient delay in presentation of rectal bleeding has been identified as a factor in delayed diagnosis among patients with colorectal cancer. The aim of this study was to identify demographic or psychological factors, or beliefs or behaviors related to delay in presentation of rectal bleeding. In 93 patients presenting with this symptom to their general practitioner, delay ranged from 0 to 249 days with a median of 7 days; 27 (29 percent) delayed more than 14 days. Delay was unrelated to age, sex, ethnic origin, competence in English, length of schooling, social status, availability of social support, measured psychologic traits, and to the belief that the cause might be cancer. The proportions delaying more than 14 days were statistically significantly elevated among those who were not worried by the bleeding (47 percent delayed); those who did not regularly look at their feces or the toilet paper after use (37 percent); and those who took some other action before presenting to their general practitioner (43 percent).

Adult↗
PubMed1990

Immunoscintigraphy of colorectal cancer with an antibody to epithelial membrane antigen (EMA).

Immunoperoxidase staining of LICR-LON M8, a mouse monoclonal antibody reactive with epithelial membrane antigen, showed a strong reaction with colorectal cancer. This finding prompted an immunoscintigraphic study of colorectal cancer patients using this antibody. Sixteen patients had external gamma scintigraphy after intravenous injection of indium 111-labeled M8. Positive scans were obtained in 11 of the 13 patients with primary colorectal cancers, and 2 of the 3 patients with recurrent tumors. The high indium 111 background in the liver prevented the detection of hepatic metastases in 5 patients. Twelve patients had samples taken of tumor, normal colon, and venous blood at the time of surgery. The ratio of labeled antibody uptake in tumor to that of blood was 5.1 (+/- 3.6 S.D.), which was significantly different (P = 0.001) to that of the similar ratio for normal colon (2.0 +/- 1.6 S.D.). The tumor to normal colon uptake ratio was 2.6 (+/- 1.3 S.D.). These results suggest a specific uptake of indium 111-labeled M8 by colorectal cancer.

Aged↗
PubMed1990

The pharmacology of N-methyl LTC4; a metabolically stable LTC4-mimetic.

N-methyl LTC4 (NMLTC4) a synthetic analogue of LTC4, has been shown not to be a substrate for gamma-glutamyl transpeptidase. NMLTC4 produced contractions of the guinea pig ileum and trachea with pD2 values of 7.7 +/- 0.12 (n = 6) and 8.1 +/- 0.1 (n = 6) respectively, compared with values of 9.0 +/- 0.1 (n = 5) and 8.0 +/- 0.2 (n = 6) for LTC4. The concentration-response curve to LTC4 and NMLTC4 on ileum was displaced to the right by FPL55712. The corresponding pA2 values were 6.3 +/- 0.3 (n = 10) for LTC4 and 5.7 +/- 0.2 (n = 6) for NMLTC4. In the presence of acivicin, a gamma-glutamyl transpeptidase inhibitor, the LTC4 concentration-response curve on trachea was displaced to the left, but the NMLTC4 curve was unaffected. The comparative potencies in the presence of acivicin on trachea indicate that LTC4 is approximately 6 times more potent than NMLTC4 whereas on ileum, in the presence of FPL55712 LTC4 is approximately 14 times more potent. In-vivo NMLTC4 is a weak bronchoconstrictor substance being 20-30 less potent than LTC4. However, unlike the in-vitro studies the bronchospasm was significantly reduced by pretreatment with LTD4 antagonists. NMLTC4 administered intravenously produced a pronounced hypertensive effect which appeared to be due to peripheral vasoconstriction.

Animals↗
PubMed1990

Regulation of a thermostable alpha-amylase of Streptomyces thermoviolaceus CUB74: maltotriose is the smallest inducer.

We have examined induction and repression by various sugars and carbon sources of the synthesis of a thermostable alpha-amylase in its natural host, S thermoviolaceus CUB74. The smallest molecule capable of inducing synthesis of the enzyme was maltotriose whereas maltose had no effect which might suggest a different control system from that found in other streptomycete amylases. Addition of mannitol to the growth medium impeded the alpha-amylase induction whereas glucose had no effect. After cloning of its gene into a new streptomycete host, S lividans TK24, the S thermoviolaceus alpha-amylase could not be induced by any of the sugars tested.

Enzyme Induction↗
PubMed1990

Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion.

We have extended the work on chymosin production in Aspergillus by constructing an expression vector in which the cDNA encoding bovine prochymosin B was fused in frame immediately following the codon for the last amino acid of the A. awamori glucoamylase (glaA) gene. Transformation of A. awamori with this plasmid led to the secretion of considerably higher amounts of chymosin than obtained with previous chymosin expression vectors. We present evidence that mature chymosin is autocatalytically released from the glucoamylase-chymosin fusion protein after secretion.

Amino Acid Sequence↗
PubMed1990

Efficient gene transfer in live mice using a unique retroviral packaging line.

Irradiated mice were transplanted with cells containing a foreign gene to evaluate gene transfer and expression as a model for gene therapy. Using a uniquely safe and efficient retroviral packaging line developed in this laboratory (GP + E86), we demonstrate efficient and safe long-term transfer of the neomycin resistance (neoR) gene into mice. By infusing cells obtained from spleen colonies of primary post-transplant mice marked with the neoR gene into irradiated recipients, secondary and tertiary generations of recipient mice were produced. There was very low reconstitution activity of single stem cells in these successive generations of mice. We conclude that many more than one stem cell is necessary for successful long-term bone marrow transplantation in mice, presumably as a result of the relatively low frequency of stem cell cycling.

Animals↗
PubMed1990

Retroviral gene transfer using safe and efficient packaging cell lines.

One of the requirements for the use of retroviral vectors in human gene therapy is a packaging cell line which is incapable of producing replication-competent virus and which produces high titers of replication-deficient vector virus. Wild-type virus may be produced through recombinational events between the helper virus and a retroviral vector. We have constructed an ecotropic packaging cell line, GP + E-86, and an amphotropic packaging cell line, GP + envAm12, in which the viral gag and pol genes are on one plasmid and the viral env gene is on another plasmid. Both plasmids contain deletions of the packaging sequence and the 3' LTR. The fragmented helper virus genomes, when introduced into 3T3 cells, produce titers of retrovirus which are comparable to the titers produced from packaging cells containing the helper virus genome on a single plasmid. We have found no evidence for the generation of wild-type retrovirus using the GP + E-86 and GP + envAm12 packaging lines, either alone or in combination with the N2 retroviral vector. We also show that these packaging cell lines can be used to transfer the neoR gene of the N2 vector into mouse hematopoietic cells, followed by successful (48-52%), long-term (up to 200 days) transplantation into irradiated recipients. These results indicate that these packaging lines are safe and efficient for use in experiments designed for murine (using GP + E-86) and human (using GP + envAm12) gene therapy.

Animals↗
PubMed1990

Chest wall motion and expiratory muscle use during phonation in normal humans.

The pattern of rib cage (RC) and abdomen (AB) motion and the electromyograms of the triangularis sterni (TS) and abdominal external oblique (EO) muscles were studied during speech and reading in six normal uninformed subjects in the sitting posture. Most phrases were started from within the tidal breathing range and extended below RC and AB spontaneous end-expiratory volumes. On the average, 75% of the change in chest wall volume occurred below the resting end-expiratory level. The expired volume resulted from a large predominance of RC displacement, and this was accompanied by marked recruitment of the TS. The EO was also generally activated, but the pattern of activation was less consistent. We conclude that 1) speech occurs primarily below the spontaneous end-expiratory level; 2) most of the volume change is caused by active emptying of the RC produced, at least in part, by contraction of the TS; 3) concomitant activation of the abdominal muscles serves to optimize the inspiratory function of the diaphragm, which has to contract rapidly between phrases to refill the respiratory system.

Adult↗
PubMed1990

A preliminary study of unresolved grief in families of seriously mentally ill patients.

Investigations of the grief reactions experienced by families when a relative develops a serious mental illness have been hampered by the lack of an appropriate instrument to measure such reactions. The authors devised the Mental Illness Version of the Texas Inventory of Grief, adapted from the Texas Revised Inventory of Grief, to assess family members' initial and present feelings about their relative's loss of mental health. A test of the instrument with 58 family members of patients with schizophrenia or bipolar disorder revealed a surprisingly low level of initial grief but higher levels of present grief.

Adaptation, Psychological↗