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Objective: To determine the existence and frequency of silent autonomic dysreflexia in subjects with a complete spinal cord injury (SCI) above the neurologic level of T6., Design: Prospective design., Setting: Blood pressure monitoring of subjects during a routine bowel program., Participants: Ten subjects with chronic (>1 y), complete (American Spinal Injury Association Impairment Scale class A) SCI with a neurologic level of injury above T6., Interventions: Not applicable., Main Outcome Measures: An increase in systolic blood pressure (SBP) of greater than 20 to 40 mmHg above baseline or an SBP greater than 150 mmHg., Results: The mean resting blood pressure for the subject group was 104/65 mmHg. During the bowel program, no subject reported experiencing any of the classic symptoms of autonomic dysreflexia. The mean maximum blood pressure recorded during the bowel program was 160/90 mmHg. All of the patients had an increase in SBP greater than 20 mmHg above baseline, and 70% had an increase in SBP greater than 40 mmHg above baseline. Sixty percent of subjects had an increase in SBP greater than 150 mmHg, with 40% of subjects reaching an SBP greater than 170 mmHg at least once during their bowel program., Conclusion: Silent autonomic dysreflexia occurs frequently in SCI during bowel programs. Further study is recommended to determine whether preventative measures or treatment is needed., (Copyright 2002 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation)

Engineering autologous adipose constructs from cell culture is a promising strategy to overcome limitations of conventional soft-tissue implants. A methodology is presented to experimentally determine and mathematically model the differentiation kinetics of in vitro 3T3-L1 preadipocyte cultures that can aid in construct design. Relative rates of morphological and interfacial events during adipogenesis were compared. Model results suggest that maturation of an intermediate multilocular phenotype was the rate-limiting step in morphological differentiation and had an intrinsic rate of 0.012 day(-1). Dislodgment of multilocular fat cells was the primary mechanism of cell loss during adipogenesis. The maximum rate of lipid droplet nucleation was predicted to precede that of coalescence by 10 days and to be three times faster. Coalescence probability was estimated to decrease from 33 to 11% for 4- and 8-microm-diameter droplets, respectively. Fluid drainage and the cytoskeleton between droplets could have impeded coalescence. The kinetic analysis suggests that droplet ripening was the dominant mechanism of lipid production. Applications of this research include engineering of an adipose construct and predicting surgical outcome of patients requiring soft-tissue augmentation.

Traditionally, emphasis has been placed on the roles of Th cells in generating and amplifying both cellular and humoral memory responses. Little is known about the potential contributions of B cell subsets to immunological memory. Resting memory B cells have generally been regarded as poor APC, attributed in part to the relative paucity of costimulatory molecules identified on their surface. We describe a novel subpopulation of human memory B cells that express CD80 in their resting state, are poised to secrete particularly large amounts of class switched Igs, and can efficiently present Ag to and activate T cells. This functionally distinct B cell subset may represent an important mechanism by which quiescent human B cells can initiate and propagate rapid and vigorous immune memory responses. Finally, these studies extend recent observations in the murine system and highlight the phenotypic and functional diversity that exists within the human B cell memory compartment.

It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.

The in vitro self-assembly of multicellular spheroids generates highly organized structures in which the three-dimensional structure and differentiated function frequently mimic that of in vivo tissues. This has led to their use in such diverse applications as tissue regeneration and drug therapy. Using Smoluchowski-like rate equations, herein we present a model of the self-aggregation of DU 145 human prostate carcinoma cells in liquid-overlay culture to elucidate some of the physical parameters affecting homotypic aggregation in attachment-dependent cells. Experimental results indicate that self-aggregation in our system is divided into three distinct phases: a transient reorganization of initial cell clusters, an active aggregation characterized by constant rate coefficients, and a ripening phase of established spheroid growth. In contrast to the diffusion-controlled aggregation previously observed for attachment-independent cells, the model suggests that active aggregation in our system is reaction-controlled. The rate equations accurately predict the aggregation kinetics of spheroids containing up to 30 cells and are dominated by spheroid adhesive potential with lesser contributions from the radius of influence. The adhesion probability increases with spheroid size so that spheroid-spheroid adhesions are a minimum of 2.5 times more likely than those of cell-cell, possibly due to the upregulation of extracellular matrix proteins and cell-adhesion molecules. The radius of influence is at least 1.5 to 3 times greater than expected for spherical geometry as a result of ellipsoidal shape and possible chemotactic or Fröhlich interactions. Brownian-type behavior was noted for spheroids larger than 30 microm in diameter, but smaller aggregates were more motile by as much as a factor of 10 for single cells. The model may improve spheroid fidelity for existing applications of spheroids and form the basis of a simple assay for quantitatively evaluating cellular metastatic potential as well as therapies that seek to alter this potential., (Copyright 2001 John Wiley & Sons, Inc.)

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system white matter. The association of the disease with MHC genes, the inflammatory white matter infiltrates, similarities with animal models, and the observation that MS can be treated with immunomodulatory and immunosuppressive therapies support the hypothesis that autoimmunity plays a major role in the disease pathology. Evidence supports activated CD4+ myelin-reactive T cells as major mediators of the disease. In addition, a renewed interest in the possible contribution of B cells to MS immunopathology has been sparked by nonhuman primate and MS pathological studies. This review focuses on the immunopathology of MS, outlining the hypothetical steps of tolerance breakdown and the molecules that play a role in the migration of autoreactive cells to the CNS. Particular focus is given to autoreactive T cells and cytokines as well as B cells and autoantibodies and their role in CNS pathogenesis in MS.

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.

Rotating-wall vessels are beneficial to tissue engineering in that the reconstituted tissue formed in these low-shear bioreactors undergoes extensive three-dimensional growth and differentiation. In the present study, bovine corneal endothelial (BCE) cells were grown in a high-aspect rotating-wall vessel (HARV) attached to collagen-coated Cytodex-3 beads as a representative monolayer culture to investigate factors during HARV cultivation which affect three-dimensional growth and protein expression. A collagen type I substratum in T-flask control cultures increased cell density of BCE cells at confluence by 40% and altered the expression of select proteins (43, 50 and 210 kDa). The low-shear environment in the HARV facilitated cell bridging between microcarrier beads to form aggregates containing upwards of 23 beads each, but it did not promote multilayer growth. A kinetic model of microcarrier aggregation was developed which indicates that the rate of aggregation between a single bead and an aggregate was nearly 10 times faster than between two aggregate and 60 times faster than between two single beads. These differences reflect changes in collision frequency and cell bridge formation. HARV cultivation altered the expression of cellular proteins (43 and 70 kDa) and matrix proteins (50, 73, 89 and 210 kDa) relative to controls perhaps due to hypoxia, fluid flow or distortion of cell shape. In addition to the insight that this work has provided into rotating-wall vessels, it could be useful in modeling aggregation in other cell systems, propagating human corneal endothelial cells for eye surgery and examining the response of endothelial cells to reduced shear.

A comprehensive physical examination of the patient with acute spinal cord injury is essential in determining the initial level of injury and is the most accurate method of prognosticating neurologic recovery. Understanding neurologic recovery helps predict ultimate functional capability and needs, and helps evaluate the effectiveness of pharmacologic and therapeutic interventions.

Objective: To quantify the incidence of swallowing deficits (dysphagia) and to identify factors that predict risk for dysphagia in the rehabilitation setting following acute traumatic spinal cord injury., Design: Retrospective case-control study., Setting: Freestanding rehabilitation hospital., Patients: Data were collected on 187 patients with acute traumatic spinal cord injury admitted for rehabilitation over a 4-year period who underwent a swallowing screen, in which 42 underwent a videofluoroscopic swallowing study (VFSS)., Main Outcome Measures: VFSS was performed on patients with suspected swallowing problems. Possible antecedents of dysphagia were recorded from the medical record including previous history of spine surgery, surgical approach and technique, tracheostomy and ventilator status, neurologic level of injury, ASIA Impairment Classification, orthosis, etiology of injury, age, and gender., Results: On admission to rehabilitation 22.5% (n = 42) of spinal cord injury patients had symptoms suggesting dysphagia. In 73.8% (n = 31) of these cases, testing confirmed dysphagia (aspiration or requiring a modified diet), while VFSS ruled out dysphagia in 26.2% (n = 11) cases. Logistic regression and other analyses revealed three significant predictors of risk for dysphagia: age (p < .028), tracheostomy and mechanical ventilation (p < .001), and spinal surgery via an anterior cervical approach (p < .016). Other variables analyzed had no relation or at best a slight relation to dysphagia. Tracheostomy at admission was the strongest predictor of dysphagia. The combination of tracheostomy at rehabilitation admission and anterior surgical approach had an extremely high rate of dysphagia (48%)., Conclusion: Swallowing abnormalities are present in a significant percentage of patients presenting to rehabilitation with acute traumatic cervical spinal cord injury. Patients with a tracheostomy appear to have a substantially increased risk of development of dysphagia, although other factors are also relevant. Risk of dysphagia should be evaluated to decrease the potential for morbidity related to swallowing abnormalities.

Objective: To determine the rate of deep vein thrombosis (DVT) newly diagnosed by duplex ultrasound in patients with acute spinal cord injury (SCI) at admission for rehabilitation., Design: Retrospective case-control study., Setting: Independent specialized spinal cord rehabilitation hospital., Patients: Data were collected from records of 189 SCI patients admitted for rehabilitation over a 1-year period who underwent a duplex scan and were not admitted with a known diagnosis of DVT., Main Outcome Measures: A DVT newly diagnosed by duplex ultrasound at rehabilitation admission., Results: Twenty-two patients (11.6%) had a newly diagnosed DVT at time of admission. Chi-square analysis found no statistically significant relationship between level of injury (tetraplegia vs paraplegia), motor complete (ASIA A and B) versus incomplete status (ASIA C and D), or cause of SCI (traumatic vs nontraumatic injury) in determining a positive or negative duplex result (chi2 = 1.709, p = .191; chi2 = 1.314, p = .252; chi2 = 3.155, p = .076; respectively). Prophylaxis for DVT decreased the risk of developing a DVT: 4.1% of patients administered prophylaxis as compared to 16.4% of patients not given prophylaxis (chi2 = 6.558, p = .01). Only 38.6% of patients transferred to rehabilitation were undergoing DVT prophylaxis., Conclusions: The prevalence of DVT in acute SCI patients at admission to rehabilitation is significant. A duplex ultrasound is an important noninvasive technique to screen patients with acute and subacute SCI for DVT on admission to the rehabilitation setting regardless of the completeness, level, or cause of the patients' injury.

In previous publications, we reported the benefits of a high-aspect rotating-wall vessel (HARV) over conventional bioreactors for insect-cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker-flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first-order term. Cell-cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 +/- 1% to 24 +/- 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell-cell interactions, waste accumulation, and mass transport. These studies may benefit insect-cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. Copyright 1999 John Wiley & Sons, Inc.

This review addresses the application of three-dimensional cultures of prostatic cells to the development of novel anti-cancer therapies. A variety of therapeutic agents to combat prostate cancer are currently under development. These include cytotoxins, differentiation agents and, more recently, genetically modified tumor vaccines. Three-dimensional cultures of prostatic cells are increasingly used in preclinical research in the design of new therapies and in the development of delivery strategies for these treatments. These tissue-like structures more realistically model the structural architecture and differentiated function of the human prostate than a cellular monolayer. In doing so, three-dimensional cultures produce an in vivo-like response to therapeutic agents. Advances in tissue engineering have improved the variety, fidelity and quantity of these prostate models. To date, they have been applied to estimate the dose of new drug therapies, evaluate drug penetration into solid tumors, assess the effectiveness of drug combinations, and develop tumor vaccines.

Objective: Traumatic spinal cord injury (SCI) affects 8,000 to 10,000 individuals per year in the United States. One of the most difficult tasks confronting the clinician is the discussion of neurologic recovery and prognosis with the patient and/or family. Our objective is to provide a guide for practitioners to accurately predict neurologic outcome in acute traumatic cervical SCI (tetraplegia)., Data Source: Published reports obtained through MEDLINE search, texts, and studies presented at national conferences., Study Selection: Peer reviewed studies, in English language, that discussed prognosis after traumatic SCI., Conclusion: A comprehensive physical examination of the acute SCI patient is essential in determining the initial level and classification of the injury and is the most accurate method to predict neurologic recovery. Other diagnostic tests, including somatosensory evoked potentials, magnetic resonance imaging, and transcranial magnetic stimulation, may be helpful in further determining outcome when used in association with the clinical examination. The understanding of neurologic recovery should help predict ultimate functional capability and potential needs.

Enoxaparin, a low molecular weight heparin, has been demonstrated to be effective in the prophylaxis and treatment of deep vein thrombosis (DVT) in the general population. This study presents an analysis of the costs of subcutaneous (SQ) enoxaparin compared with intravenous (IV) heparin. Data were gathered on six spinal cord injured (SCI) patients in an acute freestanding rehabilitation center who were initially treated with SQ enoxaparin (n=3) or IV heparin (n=3) for a proximal DVT. No patients in either group developed further clinical complications. Comparison of the total costs of each treatment was performed, including the direct cost of the drug, as well as some of the costs of administration. Although the cost of enoxaparin per unit dose is higher, the total costs of enoxaparin are slightly lower, because its labor and administration costs are less. Subcutaneous enoxaparin is a safe, cost-effective, and less labor-intensive treatment, and can be of substantial benefit in the treatment of DVT in SCI patients in the rehabilitation setting.

Objective: To determine current characteristics of bowel care practices of chronic spinal cord injury (SCI) patients., Design: Prospective interview and examination of 100 SCI patients injured for more than 1 year., Setting: Freestanding rehabilitation outpatient SCI center., Participants: One hundred chronic SCI patients., Results: The following bowel program characteristics were found: alternate-day programs were most common; most subjects performed their programs in the morning; and tetraplegic subjects performed their programs less often, used suppositories more often, required greater assistance, and took longer to complete their programs., Conclusions: Bowel dysfunction in chronic SCI need not be associated with complications in the majority of cases.

This study was conducted to further investigate the natural history of the anemia, hypoproteinemia and hypoalbuminemia commonly observed in patients with traumatic spinal cord injury (SCI). Blood samples were taken from 46 traumatic SCI patients at the time of initial admission to an acute rehabilitation hospital and again approximately one year later during a routine follow-up appointment. At initial admission, 65 percent of patients were anemic (hemoglobin < 13.0 g/dl), 86.7 percent were hypoalbuminemic (albumin < 3.2 g/dl) and 48.9 percent were hypoproteinemic (total protein < 6.5 g/dl). Deficiencies were most commonly observed in tetraplegics and in patients with complete injuries. At the time of follow-up, 6.8 percent of patients were anemic, 2.2 percent demonstrated abnormally low serum protein concentrations and a significant (p = 0.01) decrease in the incidence of each deficiency was observed. Our findings suggest that anemia and decreased serum protein concentrations, while commonly observed in the acute SCI population, are much less frequently encountered in the more chronically injured. If noted in the chronic SCI patient, these deficiencies should alert clinicians to the likelihood of a concurrent process.

A methodology is presented to culture Fall Armyworm Ovary cells in simulated micrograviy using a novel bioreactor developed by NASA, the High-Aspect Ratio Vessel. In this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture. Specifically, stationary phase in the NASA vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 x 10(7) viable cells/ml and 90%, respectively. Measurements of glucose utilization, lactate production, ammonia production, and pH change indicate that simulated microgravity had a twofold effect on cell metabolism. Fewer nutrients were consumed and fewer wastes were produced in stationary phase by as much as a factor of 4 over that achieved in shaker culture. Those nutrients that were consumed in the NASA vessel were directed along different metabolic pathways as evidenced by an extreme shift in glucose utilization from consumption to production in lag phase and a decrease in yield coefficients by one half in stationary phase. These changes reflect a reduction in hydrodynamic forces from over 1 dyne/cm2 in shaker culture to under 0.5 dyne/cm2 in the NASA vessel. These results suggest that cultivation of insect cells in simulated microgravity may reduce production costs of cell-derived biologicals by extending production time and reducing medium requirements.

Simulated microgravity within the NASA High Aspect Rotating-Wall Vessel (HARV) provides a quiescent environment to culture fragile insect cells. In this vessel, the duration of stationary and death phase for cultures of Spodoptera frugiperda cells was greatly extended over that achieved in shaker-flask controls. For both HARV and control cultures, S. frugiperda cells grew to concentrations in excess of 1 x 10(7) viable cells ml-1 with viabilities greater than 90%. In the HARV, stationary phase was maintained 9-15 days in contrast to 4-5 days in the shaker flask. Furthermore, the rate of cell death was reduced in the HARV by a factor of 20-90 relative to the control culture and was characterized with a death rate constant of 0.01-0.02 day-1. Beginning in the stationary phase and continuing in the death phase, there was a significant decrease in population size in the HARV versus an increase in the shaker flask. This phenomenon could represent cell adaptation to simulated microgravity and/or a change in the ratio of apoptotic to necrotic cells. Differences observed in this research between the HARV and its control were attributed to a reduction in hydrodynamic forces in the microgravity vessel.

The high aspect rotating-wall vessel (HARV) was recently designed by NASA to cultivate animal cells in an environment that simulates microgravity. This work examines the effects of HARV cultivation on DU 145 human prostate carcinoma cells. In the HARV, these prostate cells grew in suspension on Cytodex-3 microcarrier beads to form bead aggregates with extensive three-dimensional growth between beads and on the aggregate surface. HARV and spinner-flask control cultures of DU 145 cells had similar doubling times, but the former was characterized by a higher percentage of G(1)-phase cells, larger bead aggregates, enhanced development of filopodia and microvilli-like structures on the aggregate surface, and stronger staining for select cytoskeletal proteins (cytokeratins 8 and 18, actin, and vimentin). When compared with static controls grown in a T-flask and Transwell insert, HARV cultures grew more slowly and differences in the cell cycle and immunostaining became more pronounced. These results suggest that HARV cultivation produced a culture that was less aggressive from the perspective of proliferation, more differentiated and less pliant than any of the three control cultures examined in this work. Possible factors effecting this change are discussed including turbulence and three-dimensional growth.

In this study, we compare stress protein induction in anoxic and hyperthermicSpodoptera frugiperda cells. Anoxia transiently induces a cluster of heat shock proteins at 71 and 72 kDa. This is a subset of a larger group of stress proteins induced by heat shock. Several heat shock proteins reported in this study were previously undetected inS. frugiperda. With these additional proteins, the stress response of hyperthermicS. frugiperda closely resembles that ofDrosophila melanogaster. Prior investigations of stress protein induction during oxygen deprivation focused on mammalian cells. In sharp contrast to these cells, anoxicS. frugiperda cells neither induce glucose-regulated proteins nor suppress the heat shock family of 71/72 kDa proteins. These findings provide insight into the virtually unexplored area of stress protein induction in anoxic insect cells. In addition, they help to explain the effects of oxygen deprivation on heterologous protein yield from virally infected insect cells and to develop an oxygenregulated promoter for stably transformed insect cells.

Electron paramagnetic resonance spectroscopy is used to characterize the active site dynamics of alpha-chymotrypsin solubilized in reversed micelles. Of particular interest is the behavior of the enzyme when the micellar system is subjected to enhanced gas pressures and low temperatures. At specific thermodynamic conditions, clathrate hydrates from from the intramicellar water, reducing the micelle size and water content. Also, beyond a critical pressure, micellar instbility results. The EPR spectra under these conditions indicate that the rotational correlation times increase appreciably only when the water-to-surfactant molar ratio, W(0), is reduced to values lower than 10. The EPR characterization also reveals a remarkable resilience of the enzyme when subjected to pressure-induced changes; when returned to ambient conditions, activity and active site dynamics are fully restored. (c) 1994 John Wiley & Sons, Inc.

Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.

The structure of bovine liver glutamate dehydrogenase was examined with 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TEMPO I) and 4-((4-(chloromercurio)benzoyl) amino)-2, 2, 6, 6-tetramethyl-1-piperidinyloxy (TEMPO II). ESR spectra from TEMPO I show that enzyme structure in the vicinity of this spin label was not distorted during immobilization to a Sepharose support. Deactivation studies with pyridoxal 5'-phosphate indicate that immobilization did not expose additional binding sites to TEMPO I. Spectra from TEMPO II reveal that immobilization profoundly altered conformational change induced by alpha-ketoglutarate and suppressed that induced by GTP and NADPH. This structural investigation provides insight into the altered kinetic properties of Sepharose-immobilized glutamate dehydrogenase and suggests a fundamental difference between monomers and allosteric oligomers in their structural response to immobilization.

Substrate regulation patterns were changed by covalent binding of bovine liver glutamate dehydrogenase via primary amino groups to CNBr- and CH-activated Sepharose 4B. Lineweaver-Burk plots show that the NAD activation region changed from being abrupt to elongated when the enzyme was immobilized to either support. The elongated region contains two inflection points and resembles substrate activation of several other allosteric oligomers. Glutamate induced varying degrees of abrupt activation in immobilized glutamate dehydrogenase and inhibited the native enzyme. This activation is characterized by an activation threshold, an increase in the apparent dissociation constant, and a correlation between the apparent rate constant and the degree of activation. These three features characterize other glutamate dehydrogenase systems.