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Biomedical subjects

C Ford

Publications and source records attributed to C Ford.

At least 19 recordsLinked to original sources
PubMed1999

Management of pleural effusions in children with malignant lymphoma.

PURPOSE: The aim of this study was to determine potential problems in the diagnosis and management of children with pleural effusions and malignant lymphoma as well as the efficacy of thoracentesis. METHODS: The case histories of six children with malignant lymphoma who presented with pleural effusions were reviewed. Thoracentesis was performed using the Seldinger technique. RESULTS: Four of the children presented with symptoms and chest radiograph findings similar to pneumonia. A large mediastinal mass was present in two children. Pleural fluid analysis resulted in a definitive diagnosis of lymphoma in five of the six children. Two of the children had symptoms of reexpansion pulmonary edema after removal of pleural fluid. An empyema developed in one child after thoracotomy and chest tube placement. Reaccumulation of pleural fluid was common before initiating chemotherapy. CONCLUSIONS: Malignant pleural effusions frequently are present in children with non-Hodgkin's lymphoma. They may present with respiratory distress because of the size of the effusion, the mediastinal mass, or both. Management of these pleural effusions is associated with potential complications, some of which are life threatening. Thoracentesis is the initial diagnostic and therapeutic procedure of choice. The use of a Seldinger technique for thoracentesis has proved useful and safe. In patients with large effusions, aggressive removal of the pleural fluid may be followed by reexpansion pulmonary edema.

Adolescent↗
PubMed1999

Mutations to alter Aspergillus awamori glucoamylase selectivity. IV. Combinations of Asn20-->Cys/Ala27-->Cys, Ser30-->Pro, Gly137-->Ala, 311-4 loop, Ser411-->Ala and Ser436-->Pro.

Six previously constructed and nine newly constructed Aspergillus awamori glucoamylases with multiple mutations made by combining existing single mutations were tested for their ability to produce glucose from maltodextrins. Multiple mutations have cumulative effects on glucose yield, specific activity and thermostability. No general correlation between glucose yield and thermostability was observed, although mutations that presumably impede unfolding at high temperatures uniformly increase thermostability and generally increase glucose yield. Peak glucose yields decrease with increasing temperature. The best combination of high glucose yield, high specific activity and high thermostability occurs in Asn20-->Cys/Ala27-->Cys/Ser30-->Pro/Gly137-->Ala glucoamylase.

Aspergillus↗
PubMed1998

pEg7, a new Xenopus protein required for mitotic chromosome condensation in egg extracts.

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.

Adenosine Triphosphatases↗
PubMed1998

Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes.

In most multicellular organisms direct cell-cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

Animals↗
PubMed1998

Germline transcription and recombination of a murine VDJmudeltagamma1 transgene.

To investigate the regulation of Ig switch recombination, we have constructed mice with a 56 kb VDJmudeltagamma1 transgene. This transgene included an anti-nitrophenyl VDJ segment, Smu, Cmu, Cdelta, Igamma1, Sgamma1, Cgamma1 and the Cgamma1 membrane exons from the murine Igh(a) haplotype. Two founder lines were produced, with very similar characteristics. Transgenic B cells expressed normal amounts of Cmu (which is >95% transgenic), Cdelta and other cell surface markers, and normal amounts of VDJ and Cmu RNA. Gamma1 germline transcription of the transgenes is properly regulated since stable transcripts were not expressed in B cells treated with lipopolysaccharide (LPS) alone, nor in thymus or non-lymphoid tissues, but were expressed after treatment of B cells with LPS + IL-4 or CD40L + IL-4. B cells from both lines of transgenic mice expressed transgenic gamma1a after in vitro culture with CD40L + IL-4, but not after culture with CD40L alone. However, the CD40L + IL-4 induced IgG1 precursor frequency is much lower for VDJmudeltagamma1 transgenic B cells (1:240-760) than for non-transgenic B cells (1:9). Analysis of DNA from transgenic hybridomas indicated that switch recombination can take place in switch (S) regions, but can also take place outside S regions. These results indicate that targeting of switch recombinase to S regions must include regulation beyond the S regions themselves and correct germline transcription. This additional regulation might include cis-acting elements or appropriate spacing or arrangement of the recombining elements.

Animals↗
PubMed1998

Mutations to alter Aspergillus awamori glucoamylase selectivity. I. Tyr48Phe49-->Trp, Tyr116-->Trp, Tyr175-->Phe, Arg241-->Lys, Ser411-->Ala and Ser411-->Gly.

Glucoamylase mutations to reduce isomaltose formation from glucose condensation and thus increase glucose yield from starch hydrolysis were designed to produce minor changes in the active site at positions not totally conserved. Tyr175-->Phe and Ser411-->Gly glucoamylases had catalytic efficiencies on DP 2-7 maltooligosaccharides like those of wild-type glucoamylase, while the catalytic efficiencies of Tyr116-->Trp, Arg241-->Lys and Ser411-->Ala glucoamylases were reduced by about half and Tyr48Phe49-->Trp glucoamylase had little remaining activity. Tyr175-->Phe, Ser411-->Ala and Ser411-->Gly glucoamylases had decreased ratios of the initial rate of isomaltose formation from glucose condensation to that of glucose formation from maltodextrin hydrolysis at both 35 and 55 degrees C compared with wild-type glucoamylase. Arg241-->Lys glucoamylase had a very similar ratio, while Tyr116-->Trp glucoamylase had a higher ratio. The highest glucose yields from maltodextrin hydrolysis were by the mutant glucoamylases having the lowest ratios of initial rates of isomaltose formation to glucose formation and this predicted high glucose yields better than the ratio of catalytic efficiency for maltose hydrolysis to that for isomaltose hydrolysis.

Arginine↗
PubMed1998

Mutations to alter Aspergillus awamori glucoamylase selectivity. II. Mutation of residues 119 and 121.

Mutations Ser119-->Glu, Ser119-->Gly, Ser119-->Trp, Gly121-->Ala and Gly121-->Ala/Ser411-->Gly were constructed in glucoamylase to change substrate specificity. Mutation Ser411-->Gly was already known to decrease glucoamylase selectivity toward isomaltose formation and to increase peak glucose yield. All mutated glucoamylases had slightly lower specific activities on maltose than on wild-type glucoamylase. Ser119-->Glu, Ser119-->Gly and Ser119-->Trp glucoamylases were about as active on isomaltose and DP 4-7 maltooligosaccharides as wild-type glucoamylase. Gly121-->Ala and Gly121-->Ala/Ser411-->Gly glucoamylases were less active. At 55 degrees C Ser119-->Glu, wild-type, Ser119-->Trp, Ser119-->Gly, Gly121-->Ala and Gly121-->Ala/Ser411-->Gly glucoamylases had progressively higher peak glucose yields, generally in the opposite order to their activities. There was also an inverse correlation between peak glucose yield and ratio of initial rate of isomaltose production from glucose condensation to that of glucose production from maltodextrin hydrolysis. The effect of mutations Gly121-->Ala and Ser411-->Gly was not additive in predicting the effect of the double mutation on the ratio or on peak glucose yield.

Aspergillus↗
PubMed1998

Protein engineering of Aspergillus awamori glucoamylase to increase its pH optimum.

To increase the pH optimum of glucoamylase (GA), five mutations-S411G, S411A, S411C, S411H and S411D--were designed to destabilize the carboxylate ion form of Glu400, the catalytic base, by removing or weakening the hydrogen bond between Ser411 and Glu400, and thereby raising its pK. The substitution of alanine, histidine and aspartate were also designed to study the additional effects of polarity and both positive and negative charges, respectively. S411G GA had catalytic efficiencies like those of wild-type GA for isomaltose, maltose and maltoheptaose hydrolysis at pH 4.4, while S411A and S411C GAs had 54-74% and S411H and S411D GAs had only about 6-12% of wild-type catalytic efficiencies. All five mutations increased the pH optimum in the enzyme-substrate complex, mainly by raising pK1 values. S411A is the best performing and most industrially promising of the pH mutants isolated to date. S411A GA increased the pH optimum by 0.8 units for both maltose and maltoheptaose hydrolysis while maintaining a high level of activity and catalytic efficiency. In hydrolysis of 28% DE 10 maltodextrin, S411A GA had a pH optimum of 7 compared with pH 5.6 for wild-type GA, and had higher initial rates of glucose production than wild-type GA at all pH values tested above pH 6.6.

Aspergillus↗
PubMed1998

Mutations to alter Aspergillus awamori glucoamylase selectivity. III. Asn20-->Cys/Ala27-->Cys, Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Lys108-->Met, Gly137-->Ala, 311-314 Loop, Tyr312-->Trp and Ser436-->Pro.

Mutations Asn20-->Cys/Ala27-->Cys (SS), Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Gly137-->Ala, Tyr312-->Trp and Ser436-->Pro in Aspergillus awamori glucoamylase, along with a mutation inserting a seven-residue loop between Tyr311 and Gly314 (311-314 Loop), were made to increase glucose yield from maltodextrin hydrolysis. No active Lys108-->Met glucoamylase was found in the supernatant after being expressed from yeast. Lys108-->Arg, 311-314 Loop and Tyr312-->Trp glucoamylases have lower activities than wild-type glucoamylase; other GAs have the same or higher activities. SS and 311-314 Loop glucoamylases give one-quarter to two-thirds the relative rates of isomaltose formation from glucose compared with glucose formation from maltodextrins at 35, 45 and 55 degrees C, correlating with up to 2% higher peak glucose yields from 30% (w/v) maltodextrin hydrolysis. Conversely, Lys108-->Arg glucoamylase has relative isomaltose formation rates three times higher and glucose yields up to 4% lower than wild-type glucoamylase. Gly137-->Ala and Tyr312-->Trp glucoamylases also give high glucose yields at higher temperatures. Mutated glucoamylases that catalyze high rates of isomaltose formation give higher glucose yields from shorter than from longer maltodextrins, opposite to normal experience with more efficient glucoamylases.

Aspergillus↗
PubMed1998

Effect on thermostability and catalytic activity of introducing disulfide bonds into Aspergillus awamori glucoamylase.

Two additional disulfide bonds and three combined thermostabilizing mutations were introduced into Aspergillus awamori glucoamylase to test their effects on enzyme thermostability and catalytic properties. The single cysteine mutations N20C, A27C, T72C and A471C were made and combined to produce the double cysteine mutations N20C/ A27C and T72C/A471C. The double cysteine mutants were expressed efficiently in Saccharomyces cerevisiae, and disulfide bonds formed spontaneously after fermentation. At 50 degrees C, the single mutants N20C and A27C had decreased specific activity, whereas the specific activity of the double mutants N20C/A27C and T72C/A471C were similar to wild-type glucoamylase. The N20C/A27C mutation increased thermostability, with an increased activation free energy of 1.5 kJ/mol at 65 degrees C, while the single mutation A27C only slightly increased thermostability and N20C decreased it. The other disulfide bond-forming mutation T72C/A471C did not affect thermostability at pH 4.5. The N20C/A27C mutation was separately combined with two other thermostabilizing mutations, G137A and S436P. Thermostabilities of all of the combined mutated glucoamylases were additive. N20C/A27C/G137A glucoamylase had higher specific activity than wild-type glucoamylase from 45 to 67.5 degrees C. The disulfide bond between positions 20 and 27 connects the C-terminus of helix 1 and the following beta-turn, suggesting that this region is important for glucoamylase thermostability.

Aspergillus↗
PubMed1998

The value of screening for Down's syndrome in a socioeconomically deprived area with a high ethnic population.

OBJECTIVE: To assess the utility of biochemical antenatal screening for Down's syndrome in a socioeconomically deprived area with a high proportion of Asian women from the Indian Subcontinent. DESIGN: Audit of Down's syndrome biochemical screening service over a four-year period. SETTING: Teaching hospital and community antenatal clinic in inner city Birmingham. POPULATION: Women booked between October 1992 and December 1996. METHODS: Blood for screening was collected between 14 and 21 weeks gestation, alpha-fetoprotein and intact human chorionic gonadotrophin were measured in serum and the risk of Down's syndrome was calculated. MAIN OUTCOME MEASURES: Uptakes of screening and amniocentesis, screen positive rate, odds of being affected given a positive result, miscarriages associated with amniocentesis offered following a high risk result, detection rate, number of Down's cases prevented and a cost analysis. Outcome measures were compared between Asians and Caucasians. RESULTS: Overall 11,974 women (71%) accepted serum screening. The screen positive rate was 8.3% in Asians and 5.0% in Caucasians. The uptake of amniocentesis in women following a high risk result was 54% overall (35% Asian, 67% Caucasian). Nineteen cases of Down's syndrome were identified, of which 13 occurred in women who opted for biochemical screening. The detection rate of the biochemical screening programme was 85% (11/13). Of these 11 cases, six (none of whom were Asian) elected to have an amniocentesis, of whom four thereafter had a termination. CONCLUSION: In this study the public health benefits of screening for Down's syndrome in a socioeconomically deprived area with a high Asian population, were small.

Amniocentesis↗
PubMed1998

Maturation promoting factor activation in early amphibian embryos: temporal and spatial control.

The cytoplasmic localisation of factors capable of influencing the behaviour of nuclei has long been considered a potential mechanisms for generating cell differences during development. Yoshio Masui was instrumental in identifying two cytoplasmic factors, maturation promoting factor (MPF) and cytostatic factor (CSF), defining the first biological assay for their isolation and characterisation. These biological assays involved the transfer of cytoplasm between amphibian oocytes, MPF being able to promote meiotic maturation (progression to MII) and CSF to stabilise the MII state. Masui was subsequently involved in developing a 'cell-free' system with the potential for analysis not just of MPF and CSF, but many aspects of nucleo-cytoplasmic interaction. Masui and Markert initially showed that MPF activity could be generated in enucleate oocytes following progesterone stimulation, indicating a cytoplasmic origin. Masui subsequently showed that MPF activity was distributed unevenly through the egg of Rana pipiens during maturation. In this review we will consider the historical context in which the MPF assays were established, then briefly consider some of the molecular components that are now known to influence MPF activation. We will then consider evidence for the asymmetric activation of MPF and the possibility that the nucleus contributes to MPF activation in early embryos.

Animals↗
PubMed1997

Effect of introducing proline residues on the stability of Aspergillus awamori.

In Aspergillus awamori glucoamylase, Ala27, Ala393, Ala435, Ser436 and Ser460 were replaced with proline residues, in order to stabilize the enzyme by forming more rigid peptide backbones. Specific activities were unaffected except for a decrease in Ser460-->Pro glucoamylase. Thermostability was increased in Ser436-->Pro glucoamylase, unchanged in Ala435-->Pro glucoamylase and decreased in Ala27-->Pro, Ala393-->Pro glucoamylases. As measured by circular dichroism, mutant glucoamylases Ala435-->Pro and Ser436-->Pro resisted unfolding caused by guanidine hydrochloride at pH 4.5 and 25 degrees C better than wild-type glucoamylase, whereas mutant glucoamylases Ala27-->Pro, Ala393-->Pro and Ser460-->Pro were more susceptible to unfolding than wild-type glucoamylase, reaching a level of 50% unfolded enzyme at guanidine hydrochloride concentrations 0.50-0.75 M lower than that of the wild-type enzyme. Mutations Ala435-->Pro and Ser436-->Pro are located in a non-regular structure, which is assumed to be stabilized by these mutations. The Ala27-->Pro residue is partially buried, which may result in unfavorable steric contact and/or regional strains; mutation Ala393-->Pro results in loss of a hydrogen bond, since the N of the proline residue does not have an extra hydrogen to act as donor; and mutation Ser480-->Pro eliminates an O-glycosylation site, which could explain how these mutations destabilized glucoamylase.

Aspergillus↗
PubMed1997

Glutamine-supplemented tube feedings versus total parenteral nutrition in children receiving intensive chemotherapy.

Although enteral nutrition is generally advocated in the care of children with cancer, those patients receiving intensive chemotherapy alone or in combination with bone marrow transplantation often require total parenteral nutrition (TPN). Two patients are presented illustrating some differences between enteral and parenteral feedings in children receiving intensive chemotherapy. Nasogastric glutamine-supplemented tube feedings were well tolerated both in the hospital and at home. The cost of care for the enterally supported child was less than one third of the TPN-supported child. Although TPN appears to be beneficial in some patients with cancer, it is expensive and is associated with several significant disadvantages. Among these are an increased incidence of both gram-positive and gram-negative infections and an increased incidence of gastrointestinal symptoms. Enteral nutrition is less costly than TPN and maintains the structural and functional integrity of the intestinal mucosa. The addition of certain substrates such as glutamine, arginine and omega-3 fatty acids may improve the body's immune response as well. We hypothesize that early glutamine supplemented tube feedings in children receiving intensive chemotherapy alone or in combination with bone marrow transplantation will result in improved nutrition with fewer infections and lower cost than TPN-supplemented patients. In addition, a shorter hospital stay and improved quality of life are anticipated.

Bone Marrow Transplantation↗