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Biomedical subjects

G Rao

Publications and source records attributed to G Rao.

At least 37 records · Page 2Linked to original sources
PubMed2000

Framework for online optimization of recombinant protein expression in high-cell-density Escherichia coli cultures using GFP-fusion monitoring.

A framework for the online optimization of protein induction using green fluorescent protein (GFP)-monitoring technology was developed for high-cell-density cultivation of Escherichia coli. A simple and unstructured mathematical model was developed that described well the dynamics of cloned chloramphenicol acetyltransferase (CAT) production in E. coli JM105 was developed. A sequential quadratic programming (SQP) optimization algorithm was used to estimate model parameter values and to solve optimal open-loop control problems for piecewise control of inducer feed rates that maximize productivity. The optimal inducer feeding profile for an arabinose induction system was different from that of an isopropyl-beta-D-thiogalactopyranoside (IPTG) induction system. Also, model-based online parameter estimation and online optimization algorithms were developed to determine optimal inducer feeding rates for eventual use of a feedback signal from a GFP fluorescence probe (direct product monitoring with 95-minute time delay). Because the numerical algorithms required minimal processing time, the potential for product-based and model-based online optimal control methodology can be realized.

Algorithms↗
PubMed2000

Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility.

We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His)(6)-GFP-EK-X, where X represents chloramphenicol acetyl-transferase (CAT), human interleukin-2 (hIL-2), and organophosphorous hydrolase (OPH), respectively, (His)(6) represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein-of-interest from the fusion. The CAT and OPH fusion products ( approximately 63 kDa GFP/CAT and approximately 70 kDa GFP/OPH) were expressed at 4.85 microg/mL (19.9 microg/mg-total protein) and 1.42 microg/mL (4.2 microg/mg-total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP. In the case of hIL-2 fusion ( approximately 52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet. Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins-of-interest, was made possible by simple measurement of GFP fluorescence intensity. The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found. This could be offset by many benefits foreseen in expression and purification efficiencies.

Chloramphenicol O-Acetyltransferase↗
PubMed2000

Histologic evaluation of the inflammatory response around implanted hollow fiber membranes.

Hollow fiber membranes are enjoying widespread use as barrier materials in many implanted applications. In order to predict in vivo device behavior, it is important to understand and quantify the changes to the membrane and to the tissue immediately surrounding it that occur following implantation. We have considered a range of commercially available hollow fiber membranes for their suitability as candidates for subcutaneously implanted applications. Through analysis of excised tissue sections by light microscopy, membranes were screened at 3, 6, and 12 weeks post-implantation for the ability to maintain integrity, foreign-body reaction, and thickness of the external fibrotic capsule layer. The polysulfone microfiltration membranes and cellulose diacetate membranes investigated were found to be unsuitable owing to extensive degradation. All membranes exhibited typical foreign body reaction with fibrotic capsule formation. The thinnest capsules were observed on the regenerated cellulose microdialysis membranes and the polysulfone ultrafiltration membranes. Extensive cellular penetration into the membrane matrix of the latter was observed, but did not appear to affect the foreign body reaction. A heat-sealing method was also considered for thermoplastic membranes and found to effectively prevent cellular penetration into the lumen of the hollow fiber for the duration of the 12-week implantation.

Animals↗
PubMed2000

[Tumor surgery of the upper cervical spine].

Our own series of tumors of the upper cervical spine was analyzed retrospectively. The standard treatment strategies were reevaluated. A total of nine patients (mean age 61 years, metastasis 4, plasmocytoma 3, chordoma 1, histiocytosis 1) were treated between 1/92 and 2/99. A total of 12 operations were carried out. One-step procedures (6): Three extraoral, one transoral, one dorsal and in one case combined dorsal and extraoral tumor removal were performed. Three dorsal occipitocervical or atlantoaxial stabilizations, one ventral plating and two combined ventral plating plus dorsal three-point fixations, and four vertebral body replacements were carried out. Two-step procedures (3): three extraoral tumor removals with ventral plating plus dorsal three-point fixation, in combination with vertebral body replacement in two cases. The neurological status and the quality of life (Karnofsky performance status, pain levels) were analyzed preoperatively and at the follow-up outpatient examinations (mean follow-up: 18 months). Flexion-extension radiographs were performed at the follow-up. There was no operative mortality. The transient morbidity was 11%. The operative intervention improved the quality of life in all patients. Three patients died within 27 months of operation. Tumor resection at the upper cervical spine using individually modified surgical strategies over an approach corresponding to the tumor location, stabilization and vertebral body replacement increases significantly the time of survival and quality of life with an acceptable surgical risk for complications.

Atlanto-Axial Joint↗
PubMed2000

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase.

Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5' end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence.

Aryldialkylphosphatase↗
PubMed2000

Ratiometric oxygen sensing: detection of dual-emission ratio through a single emission filter.

A new method and device for the ratiometric measurement of oxygen concentration are presented. They are based on the use of a dual-emission oxygen-sensitive dye. The method allows the exclusion of the influence of emission overlap. The detection of the dual-emission ratio is performed using a single long-pass emission filter. The device described is simpler than the widely used lifetime instruments and could easily be a stand-alone low-cost instrument.

Coloring Agents↗
PubMed2000

Age-related decrease in the Schaffer collateral-evoked EPSP in awake, freely behaving rats.

Synaptic response size in the CA1 region of the hippocampus in aged rats is reduced for a given stimulus intensity, compared with that elicited in young rats. Consistent with the in vitro findings of reduced Schaffer collateral-evoked CA1 EPSPs in old rats, the population currents evoked to iontophoretically applied AMPA are also smaller relative to the presynaptic fiber potential amplitude. On the other hand, the size of the presynaptic fiber potential and amplitude of unitary intra-cellularly recorded EPSP responses do not change across age in the CA1 region. These electrophysiological findings are consistent with the hypothesis that old rats have fewer functional synaptic contacts per Schaffer collateral axon than do young rats. The possibility that this age change arises as a result of a differential tissue recovery response to in vitro preparation was examined in the present study. CA1 presynaptic fiber potential and EPSP amplitudes evoked by the stimulation of Schaffer collateral afferents were studied in intact, freely behaving young and old rats. We confirmed in vivo the pattern of electrophysiophysiological results previously reported in vitro and found significant correlations between the synaptic response amplitudes and the accuracy of spatial behavior in the Morris swim task. The data suggest that changes in functional connectivity of old rats may be a significant contributor to cognitive changes during aging.

Afferent Pathways↗
PubMed2000

Is there a benefit of preoperative meningioma embolization?

OBJECTIVE: To evaluate the effect of preoperative embolization of meningiomas on surgery and outcomes. METHODS: In a prospective study, 60 consecutive patients with intracranial meningiomas who were treated in two neurosurgical centers were included. In Center A, embolization was performed for none of the patients (n = 30). In Center B, 30 consecutive patients with embolized meningiomas were treated. Preoperatively, tumor size and location, neurological status, and Barthel scale score were recorded. In Center B, the extent of tumor devascularization was evaluated using angiography and postembolization magnetic resonance imaging. Intraoperatively, blood loss, the numbers of blood units transfused, and the observations of the neurosurgeon concerning hemostasis, tumor consistency, and intratumoral necrosis were recorded. Postoperatively, the neurological status and duration of hospitalization were recorded. Six months after surgery, the outcomes were assessed using the Barthel scale and neurological examinations. RESULTS: The mean tumor sizes were 22.9 cc in Center A and 29.6 cc in Center B (P > 0.1). The mean blood losses did not differ significantly (646 ml in Center A versus 636 ml in Center B; P > 0.5). However, for a subgroup of patients with subtotal devascularization (>90% of the tumor) on postembolization magnetic resonance imaging scans in Center B, blood loss was less, compared with the entire group in Center A (P < 0.05). The observations of the neurosurgeon regarding hemostasis, tumor consistency, and intratumoral necrosis did not differ significantly. There were no surgery-related deaths in either center. The rates of surgical morbidity, with permanent neurological worsening, were 20% (n = 6) in Center A and 16% (n = 5) in Center B. There was one permanent neurological deficit (3%) caused by embolization. CONCLUSION: In this preliminary study, only complete embolization had an effect on blood loss. The value of preoperative embolization for all meningiomas must be reconsidered, especially in view of the high costs and risks of embolization.

Adult↗
PubMed2000

[A study of HSP70 and NF in brain contusion timing].

The changes of HSP70 and NF after brain contusion were studied by the immunohistochemistry method. HSP70 and NF were seen at 30 min after injury around the wound area, but faded away after 12-24 h. The result suggests that these two proteins could be used to estimate brain contusion time, to diagnose brain wound and to distinguish antemortem and postmortem brain contusion.

Animals↗
PubMed1999

Insect larval expression process is optimized by generating fusions with green fluorescent protein.

The insect larvae/baculovirus protein production process was dramatically simplified by expressing fusion proteins containing green fluorescent protein (GFP) and the product-of-interest. In this case, human interleukin-2 (hIL-2) and chloramphenicol acetyl-transferase (CAT) were model products. Specifically, our fusion construct was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), the UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of the product from the fusion, and the product, hIL-2 or CAT. Both the approximately 52 kDa GFPuv/hIL-2 and approximately 63 kDa GFPuv/CAT fusions were expressed in Trichoplusia ni larvae at 9.0 microg-hIL-2 and 24.1 microg-CAT per larva, respectively. The GFP enabled clear identification of the infection process, harvest time, and more importantly, the quantity of product protein. Because the GFP served as a marker, this technique obviates the need for in-process Western analyses (during expression, separation, and purification stages). As a purification marker, GFP facilitated rapid identification of product-containing elution fractions (Cha et al., 1999b), as well as product-containing waste fractions (e.g., cell pellet). Also, because the fluorescence intensity was linear with hIL-2 and CAT, we were able to select the highest-producing larvae. That is, three fold more product was found in the brightest larva compared to the average. Finally, because the GFP is attached to the product protein and the producing larvae can be selected, the infection and production processes can be made semi-continuous or continuous, replacing the current batch process. These advantages should help to enable commercialization of larvae as expression hosts.

Animals↗
PubMed1999

Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor.

Synthesis of an operon fusion protein was investigated in batch and fed-batch cultures at high cell densities of recombinant Escherichia coli JM105 [pBAD-GFP::CAT]. Glucose-limited growth was achieved without accumulation of inhibitory byproducts allowing high cell densities (110 g L(-1) DCW) to be attained. This was believed to be the highest reported value for dry cell mass of E. coli strain JM105 expressing two recombinant proteins. Transcription of the two reporter genes, green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT), was under the control of the p(BAD) promoter of the araBAD (arabinose) operon. Each protein was independently translated via separate ribosome binding sites. CAT served as a model recombinant protein product to illustrate the noninvasive quantitative reporting ability of GFP during high cell density fermentations. Expression of GFP was monitored on-line using an intensity-based optical sensor. A linear correlation between the on-line GFP intensity and the enzymatic activity of CAT allowed for in vivo real-time quantitative monitoring of a fermentation product under conditions of high biomass concentration and high productivity.

Arabinose↗
PubMed1999

Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein.

We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 microM glucose. The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose. A modulation sensor was created by combining ANS26-GGBP with a long-lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette. Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz. Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration. These results suggest an approach to glucose sensing with simple devices.

Bacterial Proteins↗
PubMed1999

Polarization-based oxygen sensor.

A new approach to oxygen sensing based on the luminescence polarization observed from a novel type of sensor is described. The oxygen sensor consists of an oxygen-sensitive silicone film containing tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride [Ru(dpp)3Cl2] and an oxygen-insensitive film of Styryl 7 in poly(vinyl alcohol). Polarizers are used to select orthogonally polarized emission components from Ru(dpp)3Cl2 and Styryl 7. The polarization of the combined emission was found to be highly sensitive to the partial pressure of oxygen. This method of polarization sensing is generic and can be used with any fluorophore which displays an analyte-dependent change in intensity.

Fluorescence Polarization↗
PubMed1999

Green fluorescent protein as a noninvasive stress probe in resting Escherichia coli cells.

We constructed and characterized three stress probe plasmids which utilize a green fluorescent protein as a noninvasive reporter in order to elucidate Escherichia coli cellular stress responses in quiescent or resting cells. Cellular stress levels were easily detected by fusing three heat shock stress protein promoter elements, those of the heat shock transcription factor sigma32, the protease subunit ClpB, and the chaperone DnaK, to the reporter gene gfpuv. When perturbed by a chemical or physical stress (such as a heat shock, nutrient [amino acid] limitation, or addition of IPTG [isopropyl-beta-D-thiogalactopyranoside], acetic acid, ethanol, phenol, antifoam, or salt [osmotic shock]), the E. coli cells produced GFPuv, which was easily detected within the cells as emitted green fluorescence. Temporal and amplitudinal mapping of the responses was performed, and the results revealed regions where quantitative delineation of cell stress was afforded.

Blotting, Western↗
PubMed1999

Spi-1/PU.1 is a positive regulator of the Fli-1 gene involved in inhibition of erythroid differentiation in friend erythroleukemic cell lines.

Spi-1/PU.1 and Fli-1 are two members of the ETS family of transcription factors whose expression is deregulated by proviral insertion in most erythroleukemic cell lines induced by the spleen focus-forming virus (SFFV) and Friend murine leukemia virus (F-MuLV) components of the Friend viral complex, respectively. In this study, we present evidence that transcription of the Fli-1 gene is positively regulated by Spi-1/PU.1 in SFFV-transformed cell lines: (i) all SFFV-transformed cell lines expressing Spi-1/PU.1 are characterized by a specific pattern of Fli-1 gene transcripts initiated in the -200 region instead of position -400 as reported for F-MuLV-transformed cell lines; (ii) these Fli-1 transcripts initiated in the -200 region are downregulated in parallel with that of Spi-1/PU.1 during hexamethylenebisacetamide (HMBA) induced differentiation; and (iii) Fli-1 transcription is upregulated in SFFV cells lines following stable transfection of a Spi-1/PU.1 expression vector. Furthermore, we found by transient transfection assays that the -270/-41 region of the Fli-1 gene displays promoter activity which is transactivated by Spi-1/PU.1. This promoter is strictly dependent on the integrity of two highly conserved ETS DNA binding sites that bind the Spi-1/PU.1 protein in vitro. Finally, we show that transfection of constitutive or inducible Fli-1 expression vectors in SFFV-transformed cells inhibits their erythroid differentiation induced by HMBA. Overall, these data indicate that Fli-1 is a target gene of the Spi-1/PU.1 transcription factor in SFFV-transformed cell lines. We further suggest that deregulated synthesis of Fli-1 may trigger a common mechanism contributing to erythroleukemia induced by either SFFV or F-MuLV.

Animals↗
PubMed1999

Hepatitis C virus NS3/4A protease.

Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.

Antiviral Agents↗