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Biomedical subjects

J Bush

Publications and source records attributed to J Bush.

At least 19 recordsLinked to original sources
PubMed2000

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering.

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34(+)lin(-) hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well receives a single hematopoietic stem cell candidate. During an experiment lasting several days to weeks, each cell-containing well is moved sequentially and serially to a microscopic imaging system. This movement is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Biobox built on the microscopic stage. New image analysis software facilitates tracking of cell movement, recording the time of cell division, and immunophenotyping of multiple, individual, or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and may be applicable to a wide range of other systems of interest in tissue engineering.

Animals↗
PubMed2000

"It's just part of being a woman": cervical screening, the body and femininity.

This paper explores the importance of cervical screening discourses in framing women's perceptions of femininity. In-depth interviews with 35 women -- which focused on experiences of, and feelings about, cervical screening -- highlighted how medical discourses embedded within the cervical screening programme shape the feelings of normalcy and sense of obligation associated with having smear tests. With the introduction of the invitation based call and re-call programme in the UK, cervical screening has moved from an ad hoc system to a programme of mass surveillance and regulation of women's bodies. The paper highlights the ways in which cervical screening discourses were negotiated, accepted and resisted by the women interviewed. Possible theoretical explanations as to why cervical screening discourses have become important in framing femininity are discussed. The paper concludes by suggesting that the meaning and objective of mass screening programmes must be brought into question and reconsidered by feminists and those involved with the planning, implementation, research and use of screening services.

Adult↗
PubMed1999

A mutation that separates the RasG signals that regulate development and cytoskeletal function in Dictyostelium.

The expression of an activated RasG, RasG-G12T, in vegetative cells of Dictyostelium discoideium produced an alteration in cell morphology. Cells underwent a transition between an extensively flattened form that exhibited lateral membrane ruffling to a less flattened form that exhibited prominent dorsal membrane ruffling. These rasG-G12T transformants exhibited a redistribution of F-actin at the cell periphery and did not undergo the rapid contraction upon refeeding that is characteristic of wild-type cells. These results suggest a role for RasG in regulating cytoskeletal rearrangement in D. discoideum. We had shown previously that expression of rasG-G12T inhibited starvation induced aggregation (M. Khosla et al., 1996, Mol. Cell. Biol. 16, 4156-4162). rasG-G12T genes containing secondary mutations were transformed into cells to test whether the effects of rasG-G12T were transmitted through a single downstream effector. Cells expressing rasG-G12T/T35S or rasG-G12T/Y40C (secondary mutations within the effector domain) exhibited normal morphology and underwent normal aggregation, suggesting that signaling through the effector domain was required for both the morphological and the development changes induced by rasG-G12T. In contrast, cells expressing rasG-G12T/T45Q (a secondary mutation in the effector distal flanking domain) exhibited normal aggregation but a morphology indistinguishable from that of rasG-G12T transformants. This result suggests that RasG regulates developmental and cytoskeletal functions by direct interaction with more than one downstream effector.

Animals↗
PubMed1999

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering.

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34 + lin hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well can receive a single hematopoietic stem cell candidate. Sequential movement of each cell-containing well to a microscopic imaging system, serially over a several-day to several-week experiment, is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Bio-box built on the microscope stage. New image analysis software facilitates in the tracking of cell movement, recording of the time of cell division, and immunophenotyping of each of multiple individual or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and also may be applicable to a wide range of other systems of interest in tissue engineering.

Biotechnology↗
PubMed1998

Overexpression of a novel rho family GTPase, RacC, induces unusual actin-based structures and positively affects phagocytosis in Dictyostelium discoideum.

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin-rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif-containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium.

Androstadienes↗
PubMed1998

Novel HLA-A and HLA-B alleles.

Nine novel HLA-A and HLA-B alleles are described: A*2609, A*6803, A*6806, B*1539, B*1540, B*2712, B*4103, B*5109, and B*5603. Most appear to have arisen by gene conversion events. B*5603 appears to have arisen by a reciprocal recombination event joining exon 2 of a B*55/ *56 allele with exon 3 of a B*15 allele. Serologically, the antigen encoded by this allele types with broad B22- and Bw6-specific alloantisera. Also unique, the antigen encoded by B*2712 does not react with B27-specific alloantisera but does react with Bw6-specific alloantisera.

Alleles↗
PubMed1997

Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization.

Phosphatidylinositide 3-kinases (PI3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum. DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (delta ddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, delta ddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme alpha-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that delta ddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1-3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, delta ddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

Actins↗
PubMed1997

Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexposed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles, the size of postlysosomes (> 2.5 microns) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, > 90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H(+)-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.

Amino Acid Sequence↗
PubMed1996

Neurotoxin formation from pilot-scale incineration of synthetic ester turbine lubricants with a triaryl phosphate additive.

The high-temperature combustion of synthetic ester turbine engine lubricants has been performed by diluting the lubricant 5, 15, or 25% in diesel fuel and burning the mixture in a pilot-scale boiler facility. The effluent gas from this combustion system was carefully monitored for the formation of a potent neurotoxin, trimethylolpropane phosphate (TMPP). Although TMPP was not detected in the gaseous effluent, elevated levels of the neurotoxin were found in scrapings from the inside of the boiler system. Because of the extreme toxicity of this compound, significant dermal exposure could be a potential risk to incinerator operation and maintenance personnel.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed1996

A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum.

The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in mutant cells, resulting in an accumulation in acidic smaller vesicles, probably lysosomes. Also, RabD N121I-expressing cell lines missorted a small but significant percentage of newly synthesized lysosomal alpha-mannosidase precursor polypeptides. However, the majority of the newly synthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway.

Animals↗
PubMed1995

The role of food aid in drought and recovery: Oxfam's North Turkana (Kenya) drought relief programme, 1992-94.

Regional droughts carry the seeds of catastrophe: the immediate risk is famine; the long-term risk is destitution. Preventing both situations is an appropriate, if not essential, goal for relief agencies. In the past, responses to hunger in Turkana District (north-west Kenya) have taken the form of traditional feeding programmes. A better understanding of the boom/bust cycles in pastoralist systems has, however, produced new relief strategies. A central tenet of these strategies is the acceptance that relief aid should assume two roles: humanitarian--to overcome food deficits--and economic--to overcome 'income' deficits. Arguments in favour of a broader role for food aid are tested with a case study of a drought relief programme in north Turkana between 1992-1994. Field studies confirmed that when food aid is integrated as an asset into household resources, it can strengthen economic recovery. Ultimately, the extent to which relief operations are able to protect both people's lives and their livelihoods is the key to more sustainable development in drought-prone areas.

Adult↗
PubMed1994

Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits.

Highly purified lysosomes, prepared by magnetic fractionation of homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypoosmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25,000 to 100,000 were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5-diodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classified as integral membrane proteins, and were, in general, larger than 45 kDa and negatively charged due in part to the presence of mannose 6-sulfate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. Finally, based on N-terminal sequence analysis of a major 41-kDa lysosome-associated membrane protein, we cloned a cDNA that encodes a protein (DVA41) highly homologous to a yeast and a bovine vacuolar ATPase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development.

Adenosine Triphosphatases↗
PubMed1994

Molecular cloning and characterization of the full-length cDNA encoding the developmentally regulated lysosomal enzyme beta-glucosidase in Dictyostelium discoideum.

The developmentally regulated Dictyostelium discoideum lysosomal enzyme beta-glucosidase is synthesized as a membrane-associated glycosylated precursor polypeptide which undergoes at least two proteolytic cleavage events to generate a soluble mature lysosomally localized protein. To begin to analyze the mechanisms regulating the sorting of this protein and the regulation during development of the expression of the encoding gene, we have cloned and sequenced a 2.6-kilobase (kb) cDNA which contains a complete 2463-nucleotide open reading frame coding for beta-glucosidase. Conceptual translation of this open reading frame predicts a polypeptide similar in molecular mass to the primary translation product of 94 kDa that also contains the same amino acid sequences of two V8 protease derived-peptides generated from the purified beta-glucosidase enzyme. The D. discoideum enzyme contained regions highly homologous at the amino acid sequence level to both bacterial and fungal beta-glucosidases, although these regions did not overlap. A potential cleavable signal sequence was also found in the first 21 amino acids followed by a highly polar stretch of 49 amino acids which (based on amino acid sequencing of the mature beta-glucosidase) represents a pro region for this protein. This region is similar in location, size, and charge to the D. discoideum alpha-mannosidase pro-I region (Schatzle, J., Bush, J., and Cardelli, J. (1992) J. Biol. Chem. 267, 4000-4007). Several small hydrophobic stretches of amino acids were also distributed throughout the protein; however, no obvious transmembrane region(s) were identified which might explain the observed membrane association of the precursor protein. Finally, Northern blot analysis indicated that the gene encoding this enzyme was under developmental regulation. The steady state level of a 2.7-kb beta-glucosidase mRNA decreased significantly during the aggregation stage of development, from high levels during growth, and then increased in the form of a larger size 2.8-kb mRNA during the final stages of development.

Age Factors↗
PubMed1994

Attitude change amongst nursing students towards Australian aborigines.

A pre-test and post-test impact evaluation design was used to determine if attendance at a workshop conducted by Australian aboriginal health personnel improved the attitudes of nursing students towards Aborigines. A three-hour workshop attended by first year nursing students comprised an introduction, video and discussion. A self-administered questionnaire, consisting of socio-demographic background questions and attitude rating scale, was used to measure students' attitudes before and after the workshop. The Wilcoxon signed rank test and paired t-test were used to test for significant improvement in students' total attitude scores. Multiple regression analysis was conducted to determine if socio-demographic variables contributed to the changes in the students' attitude scores. Complete data were collected from 246 students. Results indicate improvement in attitude scores. Socio-demographic variables did not contribute to these scores.

Adult↗
PubMed1994

A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H(+)-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes.

In the course of screening a cDNA library for ras-related Dictyostelium discoideum genes, we cloned a 0.7 kb cDNA (rabD) encoding a putative protein that was 70% identical at the amino acid level to human Rab4. Rab4 is a small M(r) GTPase, which belongs to the Ras superfamily and functions to regulate endocytosis in mammalian cells. Southern blot analysis indicated that the rabD cDNA was encoded by a single copy gene while Northern blot analysis revealed that the rabD gene was expressed at relatively constant levels during growth and differentiation. Affinity-purified antibodies were prepared against a RabD fusion protein expressed in bacteria; the antibodies recognized a single 23 kDa polypeptide on western blots of cell extracts. Density gradient fractionation revealed that the RabD antigen co-distributed primarily with buoyant membranes rich in vacuolar protons pumps (V-H(+)-ATPases) and, to a lesser extent, with lysosomes. This result was confirmed by examining cell lines expressing an epitope-tagged version of RabD. Magnetically purified early endocytic vesicles and post-lysosomal vacuoles reacted more weakly with anti-RabD antibodies than did lysosomes. Other organelles were negative for RabD. Double-label indirect immunofluorescence microscopy revealed that RabD and the 100 kDa V-H(+)-ATPase subunit colocalized in a fine reticular network throughout the cytoplasm. This network was reminiscent of spongiomes, the tubular elements of the contractile vacuole system. Immunoelectron microscopy confirmed the presence of RabD in lysosome fractions and in the membranes rich in V-H(+)-ATPase. We conclude that a Rab4-like GTPase in D. discoideum is principally associated with the spongiomes of contractile vacuole complex.

Amino Acid Sequence↗
PubMed1994

Bringing the lab home with near-patient testing.

Near-patient testing has been shown to decrease costs while improving the quality of patient care. Technological advances are making this cost-effective element of caring more accessible.

Clinical Laboratory Techniques↗
PubMed1994

Isolation of two novel ras genes in Dictyostelium discoideum; evidence for a complex, developmentally regulated ras gene subfamily.

In Dictyostelium discoideum, three ras genes (rasD, rasG and rasB) and one ras-related gene (rap1) have been previously isolated and characterized, and the deduced amino acid sequence of their predicted protein products share at least 50% sequence identity with the human H-Ras protein. We have now cloned and characterized two additional members of the ras gene subfamily in Dictyostelium, rasC and rasS. These genes are developmentally regulated and unlike the previously isolated Dictyostelium ras genes, maximum levels of their transcripts were detected during aggregation, suggesting that the encoded proteins have distinct functions during aggregation. The rasC cDNA encodes a 189 amino acid protein that is 65% identical to the Dictyostelium RasD and RasG proteins and 56% identical to the human H-Ras protein. The predicted 194 amino acid gene product encoded by rasS is 60% identical to the Dictyostelium RasD and RasG proteins and 54% identical to the human H-Ras protein. Whereas RasD, RasG, RasB and Rap1 are totally conserved in their putative effector domains relative to H-Ras, RasC and RasS have single amino acid substitutions in their effector domains, consistent with the idea that they have unique functions. In RasC, aspartic acid-38 has been replaced by asparagine (D38N), and in RasS, isoleucine-36 has been replaced by leucine (I36L). In addition, both proteins have several differences in the effector-proximal domain, a domain which is believed to play a role in Ras target activation. In RasC, there is a single conservative amino acid change in the canonical sequence of the binding site for the Ras-specific monoclonal antibody Y13-259, and consequently, RasC is less immunoreactive with the antibody than either of the Dictyostelium RasD or RasG proteins. In contrast, RasS, which has three substitutions in the Y13-259 binding site, does not react with the Y13-259 antibody.

Amino Acid Sequence↗