Adsorptive control of water in esterification with immobilized enzymes: I. Batch reactor behavior

Reducing the influence of an undesired product in an enzymatic reaction could have a significant impact on the productivity of such systems. Here, we focus on the removal of water formed during an enzymatic esterification in a batch reactor. A commercial immobilized lipase preparation, known as Lipozyme, is used as the biocatalyst and propionic acid and isoamyl alcohol dissolved in hexane are the substrates. In this system, the water formed will partition between the catalyst and the medium. As the more polar reactants are converted into the less polar ester product, the water is partitioned more towards the biocatalyst and the accumulation of water eventually causes lower reaction rates. Addition of a strong-acid cation exchange resin in sodium form is found to control the water accumulation on the biocatalyst without stripping the essential water needed for the enzyme to function and substantial improvements in conversion are achieved. A mathematical model is developed to describe the batch reaction behavior with and without added absorbent, which successfully predicts the behavior of water and its effects.     

Cell-to-cell transfer of glial proteins to the squid giant axon: The glia- neuron protein transfer hypothesis

The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was evaluated in the giant fiber of the squid (Loligo pealei). When giant fibers are separated from their neuron cell bodies and incubated in the presence of radioactive amino acids, labeled proteins appear in the glial cells and axoplasm. Labeled axonal proteins were detected by three methods: extrusion of the axoplasm from the giant fiber, autoradiography, and perfusion of the giant fiber. This protein synthesis is completely inhibited by puromycin but is not affected by chloramphenicol. The following evidence indicates that the labeled axonal proteins are not synthesized within the axon itself. (a) The axon does not contain a significant amount of ribosomes or ribosomal RNA. (b) Isolated axoplasm did not incorporate [(3)H]leucine into proteins. (c) Injection of Rnase into the giant axon did not reduce the appearance of newly synthesized proteins in the axoplasm of the giant fiber. These findings, coupled with other evidence, have led us to conclude that the adaxonal glial cells synthesize a class of proteins which are transferred to the giant axon. Analysis of the kinetics of this phenomenon indicates that some proteins are transferred to the axon within minutes of their synthesis in the glial cells. One or more of the steps in the transfer process appear to involve Ca++, since replacement of extracellular Ca++ by either Mg++ or Co++ significantly reduces the appearance of labeled proteins in the axon. A substantial fraction of newly synthesized glial proteins, possibly as much as 40 percent, are transferred to the giant axon. These proteins are heterogeneous and range in size from 12,000 to greater than 200,000 daltons. Comparisons of the amount of amino acid incorporation in glia cells and neuron cell bodies raise the possibility that the adaxonal glial cells may provide an important source of axonal proteins which is supplemental to that provided by axonal transport from the cell body. These findings are discussed with reference to a possible trophic effect of glia on neurons and metabolic cooperation between adaxonal glia and the axon.     

SUBCELLULAR FRACTIONATION STUDIES RELATED TO THE PROCESSING OF NEUROSECRETORY PROTEINS IN APLYSIA NEURONS

Abstract— Neurosecretory cells (bag cells and R_3–14 neurons) in the abdominal ganglion of Aplysia californica were 'pulse-chased' in [³H]leucine and comparisons of the labeled protein profiles from the total cell homogenate versus a crude 'neurosecretory granule' fraction on acid-urea polyacrylamide gels were made, The data provides indirect support for the hypothesis that some of the post-translational processing of the neurosecretory proteins occurs intragranularly (L oh et al , 1975). In the case of the Bag cells the initial processing of the 29,000 daltons precursor appears to occur extragranularly, possibly in the rough endoplasmic reticulum cisternae.     

Protein release from the internal surface of the squid giant axon membrane during excitation and potassium depolarization

The proteins in the perfusate collected from intracellularly perfused squid giant axons were analyzed after being labeled with radioactive ¹²⁵I-labeled Bolton-Hunter reagent. The rate of protein release into the perfusate was found to be increased by the following electrophysiological manipulations of the axons: (1) repetitive electrical stimulation at 60 Hz in axons perfused with normal potassium fluoride-containing solution or at 0.125 Hz in axons perfused with tetraethylammonium containing solution, (2) perfusion with 4-amino-pyridine solution which induces spontaneous electrical activity in the axon, and (3) depolarization of the axon induced by raising the external potassium concentration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins released under these conditions yielded molecular weight profiles different from those of the extruded axoplasmic proteins. These observations indicate that there exists, in close association with the axonal membrane, a particular group of proteins, the solubility of which is readily affected by changes in the state of the membrane.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

Effects of stirring and sparging on cultured hybridoma cells

The response of hybridoma cells to fluid shear caused by stirring and sparging has been investigated in a 2-L turbine-agitated bioreactor. Viable cell count, lactate dehydrogenase (LDH) release, and antibody secretion were measured over the course of batch culture experiments under varied conditions of stirring and gas sparging. The effectiveness of Pluronic F68 as a protective agent in sparged cultures was also studied. Growth was found to be unaffected by stirring of the culture under surface aerated conditions, but gas sparging had a significant detrimental effect on growth and antibody production. The effect of sparging was reduced when cultures were supplemented with Pluronic at a level of 0.4% (w/v). Experimental data were analyzed through formulation of models for LDH release and antibody production. Rates of cell lysis could be estimated by correlating extracellular LDH levels through the model for LDH release. The lysis rate estimated for sparged conditions was sufficiently large to approximately account for the observed decrease in the specific growth rate of the culture. The presence of Pluronic apparently interfered with the LDH release mechanism, so precise estimation of lysis rates under these conditions was not possible. Sparging was found not to have a detrimental effect on antibody production in cultures without Pluronic added. Specific antibody production rates in cultures supplemented with Pluronic were about 25% higher than in sparged cultures without Pluronic added.     

Processing of normal and non-glycosylated forms of toad pro-opiocortin by rat intermediate (pituitary) lobe pro-opiocortin converting enzyme activity

The influence of glycosylation of a prohormone, pro-opiocortin, on its processing by intermediate (pituitary) lobe converting enzyme activity in vitro was studied. [3H]-arginine-labeled glycosylated and non-glycosylated pro-opiocortins were isolated from untreated, and tunicamycin treated toad neurointermediate lobes, respectively, after pulse-labeling in [3H]-arginine containing incubation media. These labeled precursors were then incubated at 37 degrees C in the presence of pro-opiocortin converting enzyme activity derived from rat intermediate lobe (pituitary) secretory granule lysates. The rates of conversion of the glycosylated and nonglycosylated pro-opiocortins to smaller peptide products, in vitro, were similar. Analysis of the peptide products by immunoprecipitation with ACTH and beta-endorphin antisera, and subsequent electrophoresis on acid-urea gels, indicate a comparable processing in vitro of the two forms of pro-opiocortin substrate. The only difference was that the normally glycosylated peptide products derived from glycosylated pro-opiocortin (i.e., 13K ACTH, 21K ACTH, and the 16K glycopeptide) differed in their gel electrophoretic mobilities from their counterparts derived from nonglycosylated prohormone, in a manner consistent with the absence of carbohydrate on the latter's peptides. These data show that glycosylation of the prohormone does not influence its processing in vitro by the converting enzyme activity.     

Membrane-Associated Cytoskeletal Proteins in Squid Giant Axons

Abstract: Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.     

Methods to Develop an Electronic Medical Record Phenotype Algorithm to Compare the Risk of Coronary Artery Disease across 3 Chronic Disease Cohorts

Background

Typically, algorithms to classify phenotypes using electronic medical record (EMR) data were developed to perform well in a specific patient population. There is increasing interest in analyses which can allow study of a specific outcome across different diseases. Such a study in the EMR would require an algorithm that can be applied across different patient populations. Our objectives were: (1) to develop an algorithm that would enable the study of coronary artery disease (CAD) across diverse patient populations; (2) to study the impact of adding narrative data extracted using natural language processing (NLP) in the algorithm. Additionally, we demonstrate how to implement CAD algorithm to compare risk across 3 chronic diseases in a preliminary study.

Methods and Results

We studied 3 established EMR based patient cohorts: diabetes mellitus (DM, n = 65,099), inflammatory bowel disease (IBD, n = 10,974), and rheumatoid arthritis (RA, n = 4,453) from two large academic centers. We developed a CAD algorithm using NLP in addition to structured data (e.g. ICD9 codes) in the RA cohort and validated it in the DM and IBD cohorts. The CAD algorithm using NLP in addition to structured data achieved specificity >95% with a positive predictive value (PPV) 90% in the training (RA) and validation sets (IBD and DM). The addition of NLP data improved the sensitivity for all cohorts, classifying an additional 17% of CAD subjects in IBD and 10% in DM while maintaining PPV of 90%. The algorithm classified 16,488 DM (26.1%), 457 IBD (4.2%), and 245 RA (5.0%) with CAD. In a cross-sectional analysis, CAD risk was 63% lower in RA and 68% lower in IBD compared to DM (p<0.0001) after adjusting for traditional cardiovascular risk factors.

Conclusions

We developed and validated a CAD algorithm that performed well across diverse patient populations. The addition of NLP into the CAD algorithm improved the sensitivity of the algorithm, particularly in cohorts where the prevalence of CAD was low. Preliminary data suggest that CAD risk was significantly lower in RA and IBD compared to DM.     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26Aa和cry28Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26Aa和cry28Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26Aa在仅消除cry26Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26Aa所在质粒有关。