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Biomedical subjects

J Howard

Publications and source records attributed to J Howard.

At least 55 records · Page 3Linked to original sources
PubMed1999

Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains.

Kinesin is a processive motor protein: A single molecule can walk continuously along a microtubule for several micrometers, taking hundreds of 8-nm steps without dissociating. To elucidate the biochemical and structural basis for processivity, we have engineered a heterodimeric one-headed kinesin and compared its biochemical properties to those of the wild-type two-headed molecule. Our construct retains the functionally important neck and tail domains and supports motility in high-density microtubule gliding assays, though it fails to move at the single-molecule level. We find that the ATPase rate of one-headed kinesin is 3-6 s(-1) and that detachment from the microtubule occurs at a similar rate (3 s(-1)). This establishes that one-headed kinesin usually detaches once per ATP hydrolysis cycle. Furthermore, we identify the rate-limiting step in the one-headed hydrolysis cycle as detachment from the microtubule in the ADP.P(i) state. Because the ATPase and detachment rates are roughly an order of magnitude lower than the corresponding rates for two-headed kinesin, the detachment of one head in the homodimer (in the ADP.P(i) state) must be accelerated by the other head. We hypothesize that this results from internal strain generated when the second head binds. This idea accords with a hand-over-hand model for processivity in which the release of the trailing head is contingent on the binding of the forward head. These new results, together with previously published ones, allow us to propose a pathway that defines the chemical and mechanical cycle for two-headed kinesin.

Adenosine Diphosphate↗
PubMed1999

Loss of p53 in F-MuLV induced-erythroleukemias accelerates the acquisition of mutational events that confers immortality and growth factor independence.

Erythroleukemias induced by Friend Murine Leukemia Virus (F-MuLV) involve the insertional activation of the proto-oncogene Fli-1, and the inactivation of the p53 tumor suppressor gene. While the activation of Fli-1 is an early, primary transforming event, p53 mutations are correlated with the immortalization of erythroleukemic cells in culture. In this study we have further analysed the role of p53 loss in F-MuLV induced erythroleukemias by examining the progression of this disease in p53 deficient mice. We found that p53-/- mice succumb to the disease more rapidly than p53+/+ littermates. Additionally, of the 112 tumors generated, 19 gave rise to immortal cell lines, eight of which were derived from p53-/- mice, and ten of which were from p53+/- mice. The ability of these primary tumor cells to grow in culture was associated with the complete loss of wild-type p53 in these cell lines. However, cells from many of the tumors induced in p53-/- hosts did not survive in vitro. These results suggest that the loss of p53 does not directly immortalize tumor cells. Instead, we have evidence to suggest that the loss of p53 promotes the accumulation of mutations that are required for survival in culture and that are capable of accelerating tumor progression in vivo. Indeed, mutations causing expression of the growth factor gene erythropoietin (Epo), were detected in two of seven Epo-independent cell lines from p53 deficient primary erythroleukemias. Moreover, the mechanism of activation of the Epo gene in one of these two Epo-independent cell lines involved genomic rearrangement, that is a hallmark of genetic instability. We propose that, in F-MuLV induced-erythroleukemias, p53 loss may encourage the accumulation of further mutations, subsequently conferring a growth advantage and immortality to the transformed erythroblasts.

Animals↗
PubMed1999

Kinesin takes one 8-nm step for each ATP that it hydrolyzes.

Conventional kinesin is a motor protein that moves stepwise along microtubules carrying membrane-bound organelles toward the periphery of cells. The steps are of amplitude 8.1 nm, the distance between adjacent tubulin binding sites, and are powered by the hydrolysis of ATP. We have asked: how many steps does kinesin take for each molecule of ATP that it hydrolyzes? To answer this question, the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesins adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. Division of the speed by the maximum microtubule-activated ATPase rate gave a stoichiometry of 1. 08 +/- 0.09 steps for each ATP hydrolyzed at 1 mM ATP. Therefore, under low loads in which the drag force << 1 pN, coupling between the chemical and mechanical cycles of kinesin is tight, consistent with conventional power stroke models. Our results rule out models that require two or more ATPs/step, such as some thermal ratchet models, or that propose multiple steps powered by single ATPs.

Adenosine Triphosphatases↗
PubMed1999

Characterization of the mammalian septin H5: distinct patterns of cytoskeletal and membrane association from other septin proteins.

The mechanisms controlling cytokinesis during yeast budding and animal cell fission appear quite different, yet both require members of the septin protein family. Mammalian homologs of this novel family of GTPases have been identified but little is known about their properties or functions. Using an antibody specific for the mammalian septin H5, we show that this protein is expressed at distinct levels in a variety of tissues. Tissue expression levels in different tissues did not coincide with those of the only previously characterized mammalian septin Nedd5. H5, like Nedd5, localizes to the cleavage furrow in mitotic fibroblast cells but in non-mitotic cells these proteins associate with actin filaments in different ways. Nedd5 predominantly localizes with stress fibers, but only associates with central portions of the microfilament bundles. In contrast, H5 associates with the entire length of the stress fibers and the cortical actin network. Conditions that disrupt the actin cytoskeleton also disrupt the filamentous patterns of both Nedd5 and H5, resulting in a punctate cytoplasmic pattern. Cell fractionation revealed that H5 co-fractionated with actin, while Nedd5 was predominantly restricted to the membrane fraction. Co-immunoprecipitation experiments revealed that although H5 will co-precipitate with Nedd5, the precipitation is not quantitative. Taken together, these results not only show that H5 behaves like a septin, but also demonstrate that individual septin proteins have distinct properties, suggesting that they may play different roles in cytokinesis and in other stages of the cell cycle.

3T3 Cells↗
PubMed1999

Antigen recognition.

Explore the source record for details and available documents.

Antigen Presentation↗
PubMed1999

Psychopathology, mother-child interaction, and infant development: substance-abusing mothers and their offspring.

The course of severe depressive symptoms from pregnancy to 6 months postpartum, as well as the occurrence of severe paranoid symptoms prenatally, were examined by the Millon Clinical Multiaxial Inventory I and the Beck Depression Inventory, in 78 women who were heavy, chronic cocaine users and who retained custody of their children after birth. Six months postpartum, the quality of caregiving was observed and assessed in the home, and the children were assessed on the Bayley MDI Index in the laboratory. Mothers who were depressed and paranoid prenatally, regardless of whether the depression continued to 6 months postpartum, were less sensitive in caregiving than women without severe symptoms of paranoia or depression during pregnancy or those who reported only depression that lifted by 6 months postpartum. Mothers who were depressed prenatally and continued to be depressed by 6 months postpartum, regardless of the presence or absence of paranoia, had infants who earned lower Bayley MDI scores than the offspring of women without severe psychological symptoms or women whose depression had lifted. Severe depressive symptoms during pregnancy, if they did not continue to 6 months postpartum, did not appear to adversely influence either caregiving or infant functioning.

Adult↗
PubMed1999

Kinesin's tail domain is an inhibitory regulator of the motor domain.

When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.

Adenosine Triphosphatases↗
PubMed1999

Phenotypic and functional characteristics of monocyte-derived dendritic cells from patients with myelodysplastic syndromes.

We have compared the phenotypic and functional characteristics of dendritic cells (DC) generated in vitro from the peripheral blood mononuclear fraction of myelodysplastic syndrome (MDS) patients (four refractory anaemia, four refractory anaemia with excess of blasts) with DCs generated in a similar way from eight healthy donors. After 10 d of culture in the presence of GM-CSF and IL-4, reduced numbers and percentages of DCs were obtained in MDS subjects. MDS DCs exhibited significantly lower expression of CD1a, CD54, CD80 and MHC class II molecules. Their ability to stimulate T lymphocytes in an allogeneic mixed leucocyte reaction was reduced in comparison to normal subjects. Furthermore, MDS DCs also showed a reduced receptor-mediated endocytosis as demonstrated by FITC-dextran uptake. Simultaneous fluorescence in situ hybridization (FISH) and immunophenotypic analysis demonstrated that MDS DCs have the same cytogenetic abnormality of the malignant clone. Taken together these findings indicate that, in MDS, DCs are part of the malignant clone and exhibit a deficient antigen uptake and presentation.

Dendritic Cells↗
PubMed1999

Sensitivity of domestic cat (Felis catus) sperm from normospermic versus teratospermic donors to cold-induced acrosomal damage.

Freeze-thawing cat sperm in cryoprotectant results in extensive membrane damage. To determine whether cooling alone influences sperm structure and viability, we compared the effect of cooling rate on sperm from normospermic (N; > 60% normal sperm per ejaculate) and teratospermic (T; < 40% normal sperm per ejaculate) domestic cats. Electroejaculates were divided into raw or washed (Ham's F-10 + 5% fetal calf serum) aliquots, with the latter resuspended in Ham's F-10 medium or Platz Diluent Variant Filtered without glycerol (20% egg yolk, 11% lactose). Aliquots were 1) maintained at 25 degrees C (no cooling; control), 2) cooled to 5 degrees C in a commercial refrigerator for 30 min (rapid cooling; approximately 4 degrees C/min), 3) placed in an ice slush at 0 degrees C for 10 min (ultrarapid cooling; approximately 14 degrees C/min), or 4) cooled to 0 degrees C at 0.5 degrees C/min in a programmable alcohol bath (slow cooling); and aliquots were removed every 4 degrees C. All samples then were warmed to 25 degrees C and evaluated for percentage sperm motility and the proportion of intact acrosomes using a fluorescein-conjugated peanut agglutinin stain. In both cat populations, sperm percentage motility remained unaffected (p > 0.05) immediately after exposure to low temperatures and after warming to 25 degrees C. However, the proportion of spermatozoa with intact acrosomes declined (p < 0.05) after rapid cooling ( approximately 4 degrees C/min) to 5 degrees C (N, 65.6%; T, 27.5%) or ultrarapid cooling ( approximately 14 degrees C/min) to 0 degrees C (N, 62.1%; T, 23.0%) in comparison to the control value (N, 81.5%; T, 77.5%). Transmission electron microscopy of cooled sperm revealed extensive damage to acrosomal membranes. In contrast, slow cooling (0.5 degrees C/min) to 5 degrees C maintained (p > 0.05) a high proportion of spermatozoa with intact acrosomes (N, 75.5%; T, 68.3%), which also remained similar (p > 0.05) between cat populations (N, 64.7%; T, 56.8%) through continued cooling to 0 degrees C. Results demonstrate that 1) rapid cooling of domestic cat sperm induces significant acrosomal damage without altering sperm motility, 2) spermatozoa from teratospermic males are more susceptible to cold-induced acrosomal damage than normospermic counterparts, and 3) reducing the rate of initial cooling markedly decreases sperm structural damage.

Acrosome↗
PubMed1999

Workplace-violence investigations by the California Division of Occupational Safety and Health, 1993-1996.

In response to the growing recognition of violence in the workplace, government agencies who oversee workplace safety are beginning to incorporate security issues into comprehensive safety plans. The California Division of Occupational Safety and Health began conducting inspections for violent events in 1993, and information from these security inspections was collected from January 1993 through January 1997. The inspections were examined by initiating source, industry, type of event, and citations issued and compared with distributions of known victimizations. The factors predicting whether a citation was issued were determined through logistic models. Over 200 inspections were conducted in 11 industries, with retail and health care establishments inspected most frequently. Employee complaints initiated 50.6% of events and accident reports initiated 40.1%. One third of inspections were initiated because of a fatal event, and 27.4% were initiated in response to a physical assault. Citations for security hazards were issued to 23.6% of businesses. Inspections initiated by employee complaints, in response to customer-related security issues, and involving non-fatal assaults were the most frequently cited reasons for inspection. The California Division of Occupational Safety and Health conducted security inspections in a large range of industries and for diverse issues, even though no specific security codes exist.

Accidents, Occupational↗
PubMed1999

Fli-1, an Ets-related transcription factor, regulates erythropoietin-induced erythroid proliferation and differentiation: evidence for direct transcriptional repression of the Rb gene during differentiation.

Erythropoietin (Epo) is a major regulator of erythropoiesis that alters the survival, proliferation, and differentiation of erythroid progenitor cells. The mechanism by which these events are regulated has not yet been determined. Using HB60, a newly established erythroblastic cell line, we show here that Epo-induced terminal erythroid differentiation is associated with a transient downregulation in the expression of the Ets-related transcription factor Fli-1. Constitutive expression of Fli-1 in HB60 cells, similar to retroviral insertional activation of Fli-1 observed in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia, blocks Epo-induced differentiation while promoting Epo-induced proliferation. These results suggest that Fli-1 modulates the response of erythroid cells to Epo. To understand the mechanism by which Fli-1 regulates erythropoiesis, we searched for downstream target genes whose expression is regulated by this transcription factor. Here we show that the retinoblastoma (Rb) gene, which was previously shown to be involved in the development of mature erythrocytes, contains a Fli-1 consensus binding site within its promoter. Fli-1 binds to this cryptic Ets consensus site within the Rb promoter and transcriptionally represses Rb expression. Both the expression level and the phosphorylation status of Rb are consistent with the response of HB60 cells to Epo-induced terminal differentiation. We suggest that the negative regulation of Rb by Fli-1 could be one of the critical determinants in erythroid progenitor cell differentiation that is specifically deregulated during F-MuLV-induced erythroleukemia.

Animals↗
PubMed1999

Validity of a modified shuttle test in adult cystic fibrosis.

BACKGROUND: The purpose of this study was to provide some evidence of the validity of a modified shuttle test (MST) by comparing performance on the MST with peak oxygen consumption (VO2peak) measured during a treadmill test in a group of adult patients with cystic fibrosis. METHOD: Twenty patients with stable cystic fibrosis performed a ramped maximal treadmill test (STEEP protocol) and the MST using a randomised balanced design. RESULTS: The relationship between the distance achieved on the MST and VO2peak was strong (r = 0.95, p<0.01) with 90% of the variance in VO2peak explained by the variance in MST distance. The relationship was represented by the regression equation (with 95% confidence intervals) VO2peak = 6.83 (2.85 to 10.80) + 0.028 (0.019 to 0.024) x MST distance. CONCLUSION: This study provides evidence of the construct validity of the MST as an objective measure of exercise capacity in adults with cystic fibrosis.

Adult↗
PubMed1999

Phases of alcohol problem prevention research.

We build on precedents from other health research to present a phases model of research for alcohol problem prevention that accommodates the special characteristics of this research. We propose a five-level model, in which research moves along a series of relevant continua: from basic to more and more applied research; from descriptive hypothesis-generating pilot studies to full-fledged, methodologically sophisticated, hypothesis-testing studies; from smaller to larger samples for testing; from greater to lesser control of experimental conditions; from more artificial "laboratory" environments to real-world geographically defined communities; from testing the effects of single prevention strategies to more complex studies of multiple strategies integrated into intervention systems; and from research-driven outcome studies to "demonstration" projects that evaluate the capacity of various types of communities to implement prevention programs based on prior evaluations. The five phases of research are: (1) foundational research to define and determine the prevalence of specific alcohol-involved problems, establish causal factors and processes that yield the specific problems or increase the risk of a problem, and provide the foundations for the development of effective prevention interventions; (2) developmental (preliminary effectiveness) studies to develop and test the likely effectiveness, safety, and costs of new interventions or to assess the effectiveness, safety, and costs of an existing intervention; (3) efficacy studies to determine the effects, safety, and costs of an intervention under optimal conditions of implementation (or availability or enforcement) and acceptance (or adoption at the community, organizational, or group level; or participation, compliance, or adherence at the individual level); (4) effectiveness studies of the real-world effectiveness of preventive interventions with purposeful or natural variation in implementation and acceptance; and (5) demonstration studies of the effects of interventions when widely disseminated. The proposed phases model for alcohol problem prevention research presented herein differs in significant ways from the models established by other National Institutes of Health agencies. Greater emphasis is placed on natural experiments, on methods development along the whole research continuum, on collapsing or combining research phases when appropriate, on recognizing the critical importance of behavioral parameters early as well as late in the research sequence, and on extending the research continuum to embrace diffusion and dissemination (i.e., technology transfer) studies. We also include examples of phased research in existing alcohol studies and a discussion of relevant issues, including cost, special populations, methods, and dissemination. If systematically followed, this model has the potential to contribute to wider testing and dissemination of prevention interventions of known effectiveness.

Alcohol Drinking↗
PubMed1999

Alcohol blocks TNFalpha but not other cytokine-mediated neuroprotection to NMDA.

BACKGROUND: We have investigated the effects of ethanol/alcohol (ETOH) on the pro-inflammatory CNS cytokine network that mediates neuroprotection to an excitotoxic challenge with the glutamate receptor agonist N-methyl-D-aspartic acid (NMDA). METHODS: Cultured murine cortical neurons were incubated with either TNFalpha, IL-1alpha, IL-1beta, or IL-6 in the presence or absence of 20 mM ETOH, maintained in an alcohol equilibrated humidified chamber, and the effects of these cytokines on neuronal survival after a chronic 20 hr exposure to NMDA was quantified. RESULTS: Neuroprotection induced by TNFalpha, but not IL-1alpha, IL-1beta, or IL-6, was inhibited by a concentration of alcohol (20 mM) that alone did not neuroprotect. Alcohol also affected the paracrine/autocrine induction of cytokine transcripts in neuronal cell cultures, which included enhancing the ability of TNFalpha to stimulate IL-6 transcripts. This result supports distinct cytokine-modulated neuroprotective pathways of which only TNFalpha is sensitive to low alcohol concentrations. We have shown previously that nicotine, acting through an alpha-bungarotoxin sensitive receptor, is also neuroprotective, but it too specifically abolishes TNFalpha-mediated neuroprotection. However, alcohol did not affect nicotine-induced neuroprotection. CONCLUSIONS: We suggest that the effects of low concentrations of alcohol on neuronal cytokine networks proceed through antagonism of neuroprotective pathway(s) unique to TNFalpha.

Animals↗
PubMed1998

Feline immunodeficiency virus is shed in semen from experimentally and naturally infected cats.

Although a laboratory isolate of feline immunodeficiency virus (FIV), FIV-NCSU1, has been transmitted by artificial insemination in domestic cats, transmission by naturally infected males during mating has not been reported. In order to determine whether virus shedding in semen is unique to the NCSU1 isolate, we analyzed electroejaculates from four specific-pathogen-free males infected with another laboratory strain, FIV-Petaluma, and eight random source males with naturally acquired infections. Seminal cell lysates from the cats infected with the Petaluma isolate were screened by nested polymerase chain reaction amplification for FIV gag DNA. Seminal cells and seminal plasma from these FIV-Petaluma cats were further analyzed for the presence of virus by cocultivation with a feline CD4+ T cell line and Crandell feline kidney cells. Electroejaculates from the naturally infected cats were cocultivated with the T cell line. Our results demonstrated that cell-free FIV was present in seminal plasma from two FIV-Petaluma cats and two naturally infected cats. Cell-associated seminal virus was detected in all of the FIV-Petaluma infected cats and one naturally infected cat. Secretion of viral gag p26 antigen, an indication of active viral replication, was evident in cocultures containing motile sperm purified by a swim-up procedure from a FIV-Petaluma cat. These results confirm that FIV shedding in semen is not restricted to a specific virus isolate. Furthermore, swim-up sperm from FIV-infected cats may be infectious in vitro.

Animals↗