Biomedical subjects
Publications and source records attributed to A Amanullah.
Myocardial contusion injury (MCI) is a complication of blunt thoracic trauma, which may occur at relatively low velocities. MCI may also occur from chest compressions during cardiopulmonary resuscitation. We review the clinical pathology, diagnostic tools, and treatment for MCI.
The bionconversion of indene to cis-(1S,2R)-indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using Rhodococcus, Pseudomonas putida, and Escherichia coli strains. This study reports on the application of multiparameter flow cytometry for the measurement of cytoplasmic membrane integrity and membrane depolarization as indicators of toxic effects of the substrate, product, and by-products using each of these strains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Measurements of the cytoplasmic membrane potential, cell viability, and respiratory activity provided a sensitive set of parameters to assess toxicity in the indene bioconversion and provided the basis for process improvements and strain selection. The toxic concentrations of the substrate, product, and by-products for each strain have been determined. The results show that it is possible to accumulate cis-(1S,2R)-indandiol and cis-1-amino-2-indanol up to 20 g/L without significant negative effects on cell physiology using any of the strains tested. The Gram-negative P. putida (421-5 and GM 730) and E. coli strains were more resistant to indene and the isolated chemicals of the biotransformation than the Gram-positive Rhodoccoccus I24 strain, possibly due to the presence of the outer membrane and efflux pump mechanisms. P. putida GM 730 and the E. coli TDO 123 strains responded similarly to toxic effects, and the E. coli TDO 123 strain was more resistant than the P. putida 421-5 strain. In addition to the recommendations for strain selection, the identified targets for bioprocess improvement include a combination of genetic as well as process engineering approaches.
The bioconversion of indene to cis-(1S,2R) indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using a Rhodococcus strain. This study using Rhodococcus I24 reports on the application of multiparameter flow cytometry for the measurement of cell physiological properties based on cytoplasmic membrane (CM) integrity and membrane depolarization as indicators of toxic effects of the substrate, indene. Quantification of intact polarized CM, intact depolarized CM and permeabilized CM of a large population of bacterial cells has been conducted using specific intracellular and membrane-binding fluorescent stains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Indene concentrations of up to 0.25 g/L (0.037 g indene/g dry cell weight) did not significantly (<5% compared to control) affect cell light-scattering properties, intact CM, membrane polarization, respiratory activity, or biomass growth. Between this value and 1.5 g/L (0.221 g indene/g dry cell weight), the changes in intact CM, respiratory activity and biomass growth were relatively insignificant (<5% compared to control), although dissipation of the membrane potential of a significant proportion of the cell population occurred at 0.50 g/L (0.074 g indene/g dry cell weight). At 2.5 g/L (0.368 g indene/g dry cell weight) there was a significant increase in the dead cell population, accompanied by changes in the extracellular cationic concentrations and substantial decrease in respiratory activity. The primary effect of indene toxicity was the disruption of the proton motive force across the cytoplasmic membrane which drives the formation of ATP. The disruption of the proton motive force may have been due to the measured changes in proton permeability across the membrane. In addition, indene may have directly inhibited the membrane-bound enzymes related to respiratory activity. The overall consequence of this was reduced respiratory activity and biomass growth. The cell physiological properties measured via flow cytometry are important for understanding the effects of toxicity at the cellular level which neither measurements of biomass growth or indandiol formation rates can provide since both are cell averaged measurements. The technique described here can also be used as a generic tool for measuring cell membrane properties in response to toxicity of other indene-resistant strains that may be possible to use as recombinant hosts to perform the biotransformation of indene. This study has demonstrated that flow cytometry is a powerful tool for the measurement of cell physiological properties to assess solvent toxicity on whole cell biocatalysts.
We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.
For the first time a laboratory-scale two-compartment system was used to investigate the effects of pH fluctuations consequent to large scales of operation on microorganisms. pH fluctuations can develop in production-scale fermenters as a consequence of the combined effects of poor mixing and adding concentrated reagents at the liquid surface for control of the bulk pH. Bacillus subtilis was used as a model culture since in addition to its sensitivity to dissolved oxygen levels, the production of the metabolites, acetoin and 2,3-butanediol, is sensitive to pH values between 6.5 and 7.2. The scale-down model consisted of a stirred tank reactor (STR) and a recycle loop containing a plug flow reactor (PFR), with the pH in the stirred tank being maintained at 6.5 by addition of alkali in the loop. Different residence times in the loop simulated the exposure time of fluid elements to high values of pH in the vicinity of the addition point in large bioreactors and tracer experiments were performed to characterise the residence time distribution in it. Since the culture was sensitive to dissolved oxygen, for each experiment with pH control by adding base into the PFR, equivalent experiments were conducted with pH control by addition of base into the STR, thus ensuring that any dissolved oxygen effects were common to both types of experiments. The present study indicates that although biomass concentration remained unaffected by pH variations, product formation was influenced by residence times in the PFR of 60 sec or longer. These changes in metabolism are thought to be linked to both the sensitivity of the acetoin and 2,3-butanediol-forming enzymes to pH and to the inducing effects of dissociated acetate on the acetolactate synthase enzyme.
OBJECTIVES: We evaluated a novel protocol of dual-isotope, gated single-photon emission computed tomographic (SPECT) imaging combined with low and high dose dobutamine as a single test for the characterization of various types of altered myocardial dysfunction. BACKGROUND: Myocardial perfusion tomography and echocardiography have been used separately for the assessment of myocardial viability. However, it is possible to assess perfusion, function and contractile reserve using gated SPECT imaging. METHODS: We studied 54 patients with ischemic cardiomyopathy using rest and 4 h redistribution thallium-201 imaging and dobutamine technetium-99m sestamibi SPECT imaging. The sestamibi images were acquired 1 h after infusion of the maximal tolerated dose of dobutamine and again during infusion of dobutamine at a low dose to estimate contractile reserve. Myocardial segments were defined as hibernating, stunned, remodeled or scarred. RESULTS: Severe regional dysfunction was present in 584 (54%) of 1,080 segments. Based on the combination of function and perfusion characteristics in these 584 segments, 24% (n = 140) were labeled as hibernating; 23% (n = 136) as stunned; 30% (n = 177) as remodeled; and 22% (n = 131) as scarred. Contractile reserve, represented by improvement in wall motion/thickening by low dose dobutamine, was observed in 83% of stunned, 59% of hibernating, 35% of remodeled and 13% of scarred myocardial segments (p<0.05). CONCLUSIONS: It is possible with this new imaging technique to characterize dysfunctional myocardium as stunned, hibernating, remodeled and nonviable. These subtypes often coexist in the same patient.
The transcription factor E2F-1 has been postulated to play a crucial role in the control of cell cycle progression because of its ability to be bound and regulated by the retinoblastoma gene product (pRb). Exogenous expression of E2F-1, under growth restrictive conditions, was shown to result in p53-dependent programmed cell death. The consequences of deregulated expression of E2F-1 on terminal differentiation of hematopoietic cells in the absence of E2F-1-mediated apoptosis, as well as mechanistic insights into how deregulated E2F-1 may affect terminal differentiation, have not been established. The autonomously proliferating M1 myeloblastic leukemia cell line, which is null for p53 expression and can be induced by interleukin-6 (IL-6) to undergo terminal macrophage differentiation with concomitant loss of leukemogenicity, provides a particularly attractive model system to address these issues. Deregulated and continued expression of E2F-1 blocked the IL-6-induced terminal differentiation program at an early blast stage, giving rise to immature cells, which continued to proliferate without undergoing apoptosis and retained their leukemogenic phenotype. Although E2F-1 blocked IL-6-mediated terminal differentiation and its associated growth arrest, it did not prevent the rapid induction of both p15(INK4B) and p16(INK4A), inhibition of cdk4 kinase activity, and subsequent hypophosphorylation of pRb. The results obtained imply that genetic alterations that both impair p53 function and deregulate E2F-1 expression may render hematopoietic cells refractory to the induction of differentiation and are, thereby, likely to play a major role in the progression of leukemias. (Blood. 2000;96:475-482)
Previously we have shown that deregulated expression of c-myc in M1 myeloid leukemic cells blocked IL-6-induced differentiation and its associated growth arrest; however, the cells proliferated at a significantly reduced rate compared to untreated cells. The basis for the increased doubling time of IL-6-treated M1myc cells was found to be due to the induction of a p53-independent apoptotic pathway. The apoptotic response was not completely penetrant; in the same population of cells both proliferation and apoptosis were continuously ongoing. Down-regulation of Bcl-2 was insufficient to account for the apoptotic response, since deregulated expression of Bcl-2 delayed, but did not block, the onset of apoptosis. Furthermore, our results indicated that the IL-6-induced partial hypophosphorylation of the retinoblastoma gene product (Rb), observed in M1myc cells, was not responsible for the apoptotic response. Finally, the findings in M1 cells were extended to myeloid cells derived from the bone marrow of wild type and p53-deficient mice, where the deregulated expression of c-myc was also shown to block terminal differentiation and induce apoptosis independent of p53. These findings provide new insights into how myc participates in the neoplastic process, and how additional mutations can promote more aggressive tumors. Oncogene (2000) 19, 2967 - 2977
Given the impact of mycelial morphology on fermentation performance, it is important to understand the factors that influence it, including agitation-induced fragmentation. The successful application of the energy dissipation/circulation function (EDC) to correlate fragmentation of Penicillium chrysogenum with agitation intensity and with different impeller types [5] has already been demonstrated. The EDC function takes into account the specific energy dissipation rate in the impeller swept volume and the frequency of mycelial circulation through that volume. In order to explore whether the EDC function can be used more generally to correlate fragmentation of different filamentous species, the present study extended the concept to agitation-induced, off-line fragmentation of Aspergillus oryzae grown in chemostat culture. The work shows that at EDC values off-line greater than that in the chemostat, fragmentation with different impellers can be correlated with the EDC. For EDC values less than those used in the chemostat, fragmentation did not occur. The earlier results of Jüsten et al. [5] with Penicillium chrysogenum are also reconsidered and found to behave similarly.
The effects of agitation on fragmentation of a recombinant strain of Aspergillus oryzae and its consequential effects on protein production have been investigated. Constant mass, 5.3-L chemostat cultures at a dilution rate of 0.05 h-1 and a dissolved oxygen level of 75% air saturation, have been conducted at 550, 700, and 1000 rpm. These agitation speeds were chosen to cover a range of specific power inputs (2.2 to 12 kW m-3) from realistic industrial levels to much higher values. The use of a constant mass chemostat linked to a gas blender allowed variation of agitation speed and hence gas hold-up without affecting the dilution rate or the concentration of dissolved oxygen. The morphology of both the freely dispersed mycelia and clumps was characterized using image analysis. Statistical analysis showed that it was possible to obtain steady states with respect to morphology. The mean projected area at each steady state under growing conditions correlated well with the 'energy dissipation/circulation" function, [P/(kD3tc)], where P is the power input, D the impeller diameter, tc the mean circulation time, and k is a geometric constant for a given impeller. Rapid transients of morphological parameters in response to a speed change from 1000 to 550 rpm probably resulted from aggregation. Protein production (alpha-amylase and amyloglucosidase) was found to be independent of agitation speed in the range 550 to 1000 rpm (P/V = 2.2 and 12.6 kW m-3, respectively), although significant changes in mycelial morphology could be measured for similar changes in agitation conditions. This suggests that mycelial morphology does not directly affect protein production (at a constant dilution rate and, therefore, specific growth rate). An understanding of how agitation affects mycelial morphology and productivity would be valuable in optimizing the design and operation of large-scale fungal fermentations for the production of recombinant proteins. Copyright 1999 John Wiley & Sons, Inc.
Agitation speed affects both the extent of motion in Xanthan fermentation broths because of their rheological complexity and the rate of oxygen transfer. The combination of these two effects causes the dissolved oxygen concentration and its spatial uniformity also to change with agitator speed. Separating these complex interactions has been achieved in this study in the following way. First, the influence of agitation speeds of 500 and 1000 rpm has been investigated at a constant nonlimiting dissolved oxygen concentration of 20% of air saturation using gas blending. Under these controlled dissolved oxygen conditions, the results demonstrate that the biological performance of the culture was independent of agitation speed as long as broth homogeneity could be ensured. With the development of increasing rheological complexity lending to stagnant regions at Xanthan concentrations >20 g/L, it is shown that the superior bulk mixing achieved at 1000 rpm, compared with 500 rpm, leading to an increased proportion of the cells in the fermentor to be metabolically active and hence higher microbial oxygen uptake rates, was responsible for the enhanced performance. Second, the effects of varying dissolved oxygen are compared with a control in each case with an agitator speed of 1000 rpm to ensure full motion, but with a fixed, nonlimiting dissolved oxygen of 20% air saturation. The specific oxygen uptake rate of the culture in the exponential phase, determined using steady-state gas analysis data, was found to be independent of dissolved oxygen above 6% air saturation, whereas the specific growth rate of the culture was not influenced by dissolved oxygen, even at levels as low as 3%, although a decrease in Xanthan production rate could be measured. In the production phase, the critical oxygen level was determined to be 6% to 10%, so that, below this value, both specific Xanthan production rate as well as specific oxygen uptake rate decreased significantly. In addition, it is shown that the dynamic method of oxygen uptake determination is unsuitable even for moderately viscous Xanthan broths. Copyright 1998 John Wiley & Sons, Inc.
The rheological complexity of Xanthan fermentations presents an interesting problem from a mixing viewpoint, because the phenomena of poor bulk blending and low oxygen mass transfer rates inherent in highly viscous fermentations (and their consequences) can be systematically investigated, even at the pilot plant scale. This study in a 150 L fermentor compares the physical and biological performance of four pairs of impellers: a standard Rushton turbine, a large diameter Rushton turbine, a Prochem Maxflo T, and a Scaba 6SRGT. Accurate in-fermentor power measurements, essential for the comparison of impellers in relation to operating costs are also reported. It is demonstrated that the agitator performance in Xanthan fermentations is very specific and the choice of which impeller to use in bioreactors to obtain enhanced performance is dependant on the applied criterion. None of the criterion favored the use of the standard Rushton turbine, therefore suggesting that there are strong grounds for retrofitting these impellers with either large diameter impellers of similar design or with novel agitators. In addition, fluid dynamic modeling of cavern formation has clearly highlighted the importance of a well mixed and oxygenated region for providing the capacity for high microbial oxygen uptake rates which govern Xanthan productivity and quality. Copyright 1998 John Wiley & Sons, Inc.
This paper is the fourth in a series aimed at improving the understanding and operation of conventional agitated fermenters for the production of the commercially important gum, Xanthan. In the first, reproducible fermentations were established and this protocol was used in studies of different agitator types and of bulk mixing and dissolved oxygen concentration in the next two. Here, building on the previous work, the influence of different glucose feeding strategies on Xanthan production in a 20-L agitated fermenter under equivalent conditions of agitation and dissolved oxygen is reported. The biological performances in three types of fed-batch cultures (a two-step glucose addition, multiple glucose-pulse feeding and continuous feeding of glucose) are compared to two batch fermentations with different initial glucose concentrations. The work confirmed that improved performance cannot be achieved by increasing the initial glucose concentration above 50 g/L nor by a single 10 g/L pulse addition (initial glucose concentration of 40 g/L) while significant nitrogen is still present. On the other hand, the simple pulse and continuous feeding strategies, after nitrogen has been essentially exhausted and under conditions of nonlimiting dissolved oxygen and similar bulk mixing, can result in a greatly enhanced performance compared to batch fermentations. Using the final Xanthan gum concentration, the yield on glucose and the overall productivity as performance indices, values of 62 g/L, 0.82 g of Xanthan/g of glucose, and 0.72 g/(L.h), respectively, were obtained compared to literature values for conventional stirred bioreactors of 15-30 g/L, 0.27-0.86 g of Xanthan/g of glucose, and 0. 12-0.43 g/(L.h).
We examined 1,159 consecutive patients who underwent adenosine stress dual isotope single-photon emission computed tomography (SPECT) and had follow-up performed at a mean of 27.5 +/- 9.1 months (94% complete) for hard events (cardiac death and myocardial infarction) and referral to cardiac catheterization after nuclear testing. During follow-up, 120 hard events occurred (11.0% hard event rate; 72 cardiac deaths [6.7% cardiac death rate] and 57 myocardial infarctions [5.3% myocardial infarction rate]). Cox proportional hazards analysis revealed that nuclear testing added incremental value after adjusting for clinical and historical variables (global chi-square increased 13 to 98 for cardiac death as the end point, global chi-square increased 19 to 105 for hard events as the end point; p <0.0001 for both). Kaplan-Meier analysis demonstrated that after clinical risk stratification of the patient population, the results of nuclear testing were further able to significantly stratify both low- and intermediate- to high-risk patients. Patients with both normal and mildly abnormal scans were at low risk of cardiac death (<1% cardiac death per year of follow-up) and the risk of events increased significantly with worsening scan result. Multivariable analysis revealed that the only predictor of referral to catheterization was the extent and severity of reversible defect present on the scan. Referral rates to early catheterization were very low in patients with normal scans and increased significantly as a function of worsening scan results. In patients who underwent myocardial perfusion SPECT using adenosine stress, the results of nuclear testing yielded incremental prognostic information and clinically relevant risk stratification. Referring physicians predominantly utilized nuclear information when referring patients to catheterization after nuclear testing and do so at rates comparable with those after exercise SPECT despite the higher risk of events in patients undergoing pharmacologic stress.
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Analysis of retroviral integration sites in MuLV-induced promonocytic leukemias has determined that two genetic loci, c-myb and Mml1, can contribute to disease development but not in the same leukemia. Recent studies aimed at understanding the function of Myb in leukemia development have focused on the consequences of ectopic Myb expression on monocytic and granulocytic differentiation in vitro. In all instances Myb was shown to block growth arrest but not commitment to differentiation, a result which is consistent with observed effects of Myb in leukemia development. No effect of Myb protein truncation was observed in these studies although similar truncations are produced as a result of insertional mutagenesis. Common integration site, Mml1, was recently identified and mapped to mouse chromosome 10 within 1cM of c-myb. Despite its linkage to c-myb, Myb mRNA and protein expression appear to be unaffected in leukemias with Mml1 integrations.
B-Myb is a transcriptional regulator of gene expression and is highly homologous to c-Myb in its N-terminal DNA binding domain. However, unlike c-myb, whose expression is restricted largely to immature hematopoietic cells, B-myb mRNA has been found to be expressed in all proliferating mammalian cell lines and is clearly regulated in a cell cycle dependent manner. That c-Myb and B-Myb proteins perform different roles in proliferation and/or differentiation is suggested by the redundancy of their expression. It was previously shown that degenerated c-Myb expression can inhibit IL-6 induced terminal differentiation of the leukemia cell line M1. We found that, unlike the downregulation of c-Myb protein which is an early response of progenitor M1 cells to IL-6 treatment, the downregulation of B-Myb occurs late, just prior to terminal differentiation and growth arrest. It was, therefore, of interest to examine the role of the murine B-Myb protein in the proliferation and differentiation of the M1 cells and to compare these effects to those of c-Myb in the same system. Clones ectopically producing B-Myb, like those ectopically expressing c-Myb, proliferated in the presence of the differentiation-inducing agent and did not undergo the programmed cell death which normally follows terminal macrophage differentiation. In addition, the cell-cycle distribution of M1/B-Myb cells was comparable to untreated cells. Although M1/B-Myb and M1/c-Myb clones treated with IL-6 appeared quite immature, differentiation markers were demonstrated to be maintained at near normal levels (e.g. MyD88, Mac-2), or be partially reduced in expression (C3, Fc and Mac-1 receptors) suggesting that the cells had undergone commitment to maturation, but were unable to terminally differentiate.
Studies have focused on two genetic loci, c-myb and Mml1, whose activation by retroviral insertional mutagenesis contribute to promonocytic leukemia in our acute monocytic leukemia (AMoL) model. Multiple mechanisms of activation of c-myb by retroviral insertional mutagenesis implicate both transcriptional deregulation and protein truncation in conversion of this proto-oncogene to an oncogene. Because transformation by c-Myb can be viewed as a block to differentiation our studies moved into two in vitro systems to evaluate effects of truncated forms of c-Myb on cytokine induced maturation of myeloid progenitors to the granulocyte and macrophage lineages. Deregulated expression of truncated and full length c-Myb did not result in maintenance of the myelomonocytic progenitor state but rather a block in differentiation at intermediate to late steps in the maturation processes of myelomonocytic cells. Our results argue that inhibition of differentiation is due to c-Myb's ability to maintain the proliferative state of cells. Interestingly, the phenotype of continuously proliferating monocytic cells resembles that of the tumor cell phenotype. Recently we identified a new target of integration, Mml1, which is rearranged in ten promonocytic leukemias that do not have c-myb rearrangements. This locus which was mapped to chromosome 10 is presently being characterized.