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Biomedical subjects

J M Lee

Publications and source records attributed to J M Lee.

At least 73 records · Page 4Linked to original sources

The production of foreign proteins from genetically modified plant cells.

While traditionally used to produce natural products, plant suspension cultures can also be utilized for the production of foreign proteins. Production of these high-value products in plant cells is an economically viable alternative to other systems, particularly in cases where the protein must be biologically active. There are several advantages to using plant cells for the large-scale production of secreted proteins. Plant cell media are composed of simple sugars and salts and are therefore less expensive and complex than mammalian media. Consequently, purification of secreted protein is simpler and more economical. Additionally, plant cell derived proteins are likely to be safer than those derived from other systems, since plant cell pathogens are not harmful to humans. In this chapter, we will review foreign protein production from plant cells. To begin, we will discuss the behavior of plant cell cultures, products produced by plant cells, protein secretion and its relationship to purification, and the performance of plant cells as compared to whole plants and other alternative hosts. After a brief discussion of gene transfer techniques, we will present strategies to overcome the limitations of protein production, including protein stabilization, novel production schemes, modeling, and scale-up considerations. To conclude, we will discuss implications for future development of this technology.

Animals↗

Microscopic and macroscopic approaches of Cu(II) removal by FSM-16.

The removal of Cu(II) by a mesoporous material, FSM-16, was studied using electron paramagnetic resonance (EPR) spectroscopy and surface complexation modeling (SCM). Free copper ions, adsorbed and precipitated Cu(II) species were qualitatively identified by in situ EPR spectroscopy of Cu-FSM-16 suspensions at room temperature and at 77 K. In addition, the adsorbed species was identified as a Cu(II) species with an axial symmetry from an analysis of the EPR spectra of "dry" Cu-FSM-16 at 77 K. On the basis of the EPR results, the removal of Cu(II) as a function of pH under various experimental conditions was successfully simulated by assuming two removal mechanisms such as surface complexation and surface precipitation. In the acidic pH range (< pH 6), free copper ions were predominant, and surface complexed then surface precipitated species became dominant as the pH increased.

Adsorption↗

Depletion of glial cell line-derived neurotrophic factor in substantia nigra neurons of Parkinson's disease brain.

The distribution of nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in substantia nigra pars compacta (SNc) of Parkinson's disease (PD) brains was investigated by immunofluorescence. Cases studied included four 69-77 year old neurologically normal male controls and four 72-79 year old male PD patients. Integrated optical densities (IODs) of immunofluorescence over individual neuromelanin-containing neurons and in areas of neuropil and the number of neurons on H & E stained adjacent sections were quantitated with the use of the BioQuant Image Analyzer. Data were statistically analyzed by ANOVA, including the unpaired two-tailed Student t-test and the Mann-Whitney test. The results showed 55.8% (P<0.0001) dropout of SNc neurons in PD brains compared to age-matched controls. Despite considerable neuronal dropout, immunofluorescent NTFs in the PD brains showed differential reductions that were consistent within the group as compared to age-matched controls: reductions were GDNF, 19.4%/neuron (P<0.0001), 20.2%/neuropil (P<0.0001); CNTF, 11.1%/neuron (P<0.0001), 9.4%/neuropil (P<0.0001); BDNF, 8.6%/neuron (P<0.0001), 2.5%/neuropil. NGF, NT-3 and NT-4 showed no significant differences within surviving neurons or neuropil. Since the depletion of GDNF both within surviving neurons and neuropil was twice as great as that of CNTF and BDNF and since the other NTFs showed no changes, GDNF, of the tested NTFs, is probably the most susceptible and the earliest to decrease in the surviving neurons of SNc. These observations suggest a role for decreased availability of GDNF in the process of SNc neurodegeneration in PD.

Aged↗

Fluoroscopic-guided covered metallic stent placement for gastric outlet obstruction and post-operative gastroenterostomy anastomotic stricture.

AIM: To evaluate the feasibility and safety of fluoroscopic-guided covered metallic stent placements in providing palliative care for patients with inoperable malignant gastric outlet obstruction. MATERIALS AND METHODS: Under fluoroscopic guidance, placement of self-expandable, covered stents was attempted in 20 patients with inoperable or recurrent gastric cancer (age range 36-79 years). All patients had inoperable gastric outlet obstruction, 13 with native anatomy and seven with post-operative anatomy (gastrointestinal anastomotic sites). All patients had intolerance to oral alimentation and/or vomiting after ingestion. Success was defined both technically and clinically. RESULTS: The placement of the stent was technically successful in 18 patients and failed in two patients (technical success: 90%). The cause of the technical failures was an inability to negotiate the guide wire through the obstruction sites in spite of the use of both fluoroscopic and endoscopic guidance. After stent placement, 15 patients were able to ingest at least liquids and had a markedly decreased incidence of vomiting (clinical success: 75%). During the mean follow-up of 6 weeks, there have been no stent reocclusion and no life-threatening complications except migration of two stents in one patient. CONCLUSION: Fluoroscopically guided covered metallic stent placement appears to be valuable for the palliative treatment of malignant obstruction of gastric outlet and post-operative gastrointestinal anastomoses.

Adenocarcinoma↗

Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver.

The hepatic expression of the ATP-dependent conjugate export pump multidrug resistance-associated protein 2 (Mrp2) is diminished in experimentally induced models of cholestasis. In this study we have examined the localization and expression of Mrp3, another member of the multidrug resistance-associated protein family, in normal liver and after obstructive cholestasis in the rat. Indirect immunofluorescence and confocal microscopy were used to determine the tissue localization and Western blot analysis was performed to quantitate the expression. In normal rat liver Mrp3 was found on the basolateral membrane of cholangiocytes and a single layer of hepatocytes surrounding the central vein. Three and 7 days after bile duct ligation Mrp3 expression was significantly increased, predominantly in hepatocytes in the pericentral region. By 14 days all hepatocytes showed basolateral membrane labeling for Mrp3 at a time when apical Mrp2 staining was significantly diminished. Proliferating bile ducts continued to stain positive, although the intensity of staining did not seem to vary. After 14 days Western blot quantitation showed that Mrp3 had increased approximately 30-fold in total liver membranes. Quantitation of Mrp3 in membranes from isolated hepatocytes of livers of sham and common bile duct-ligated (CBDL) animals showed a significant up-regulation beginning at 1 day and continuing to increase through 14 days postligation. This was in contrast to the progressive decrease in Mrp2 protein. Because Mrp3 is capable of transporting toxic bile acids, up-regulation of Mrp3 may compensate for the down-regulation of Mrp2 in obstructive cholestasis.

Animals↗

In vitro assessment of the mechanical properties of latex and non-latex orthodontic elastics.

The increased incidence of latex allergies has led to the development of non-latex orthodontic products. The purpose of this study was to compare the mechanical properties of latex and non-latex orthodontic elastics. The elastics tested were from 2 manufacturers (GAC and Masel) and made of 2 materials (latex and non-latex). The properties tested included cross-sectional area, breaking force, peak load, peak stress, stiffness, modulus, hysteresis, and 24-hour load relaxation. The data were analyzed with 1-sample t tests, an analysis of variance, and the Fisher PLSD test. Between GAC and Masel, the GAC non-latex elastics had greater breaking forces than did the Masel non-latex elastics. The Masel latex elastics had greater breaking forces than did the Masel non-latex elastics. The GAC non-latex elastics showed more viscoelastic properties than both the GAC latex elastics and the Masel non-latex elastics; however, the Masel elastics were more viscoelastic than the GAC elastics. Forces generated by the elastics decreased over 24 hours to an average load approximating 75% of the manufacturers' values (GAC latex, Masel latex, and Masel non-latex elastics) and to 60% for the GAC non-latex elastics. The mechanical properties of the non-latex elastics were not comparable to those of the latex elastics. Therefore, the clinical choice of elastics should be based on the patient's medical history and the specific mechanical properties of the type of elastic.

Analysis of Variance↗

Variable site of oropharyngeal narrowing and regional variations of oropharyngeal collapsibility among snoring patients during wakefulness and sleep.

The oropharynx shows rhythmic caliber changes in accordance with the respiratory cycle. Dynamic imaging of the oropharynx is required to evaluate airway changes associated with snoring and obstructive sleep apnea. The purpose of this study was to determine the sites of narrowing/obstruction and to measure the regional collapsibility of the oropharynx during respiration during the sleep and awake cycles in snoring patients. Seven snoring patients were included for this study. Electron Beam Tomograms of the oropharynx were taken while the patient was awake and asleep, and dynamic views were reconstructed. The site of narrowing or obstruction varied widely among patients and differed from awake to asleep. The mean of smallest minimum cross-sectional area of the oropharynx was 60.38+/-42.97 mm2 in the awake state and 16.90+/-20.43 mm2 in the asleep state. Although the collapsibility of the oropharynx showed considerable regional variations, it increased during sleep at the overall oropharynx. The low retropalatal region showed the smallest minimum cross-sectional area and the highest collapsibility; however, the most prominent changes in the minimum cross-sectional area and collapsibility during sleep were observed in the high retroglossal region.

Adult↗

A gene expression profile of Alzheimer's disease.

Postmortem analysis of brains of patients with Alzheimer's disease (AD) has led to diverse theories about the causes of the pathology, suggesting that this complex disease involves multiple physiological changes. In an effort to better understand the variety and integration of these changes, we generated a gene expression profile for AD brain. Comparing affected and unaffected brain regions in nine controls and six AD cases, we showed that 118 of the 7050 sequences on a broadly representative cDNA microarray were differentially expressed in the amygdala and cingulate cortex, two regions affected early in the disease. The identity of these genes suggests the most prominent upregulated physiological correlates of pathology involve chronic inflammation, cell adhesion, cell proliferation, and protein synthesis (31 upregulated genes). Conversely, downregulated correlates of pathology involve signal transduction, energy metabolism, stress response, synaptic vesicle synthesis and function, calcium binding, and cytoskeleton (87 downregulated genes). The results support several separate theories of the causes of AD pathology, as well as add to the list of genes associated with AD. In addition, approximately 10 genes of unknown function were found to correlate with the pathology.

Alzheimer Disease↗

Serological survey for Ehrlichia canis in urban dogs from the major population centres of northern Australia.

OBJECTIVE: To detect evidence of Ehrlichia canis infection of dogs from the major population centres of northern Australia, if present. DESIGN: Serological investigation for E. canis. PROCEDURE: The sera of 316 domestic dogs, collected from the northern Australian population centres of Townsville, Cairns, Darwin, Kununurra and Broome from May 1997 to August 1999, were investigated for evidence of infection with E. canis. Samples were tested for antibodies to E. canis using an indirect fluorescent antibody (IFA) test. The buffy coats from blood of dogs whose serum reacted in the IFA test were subsequently tested with a nested PCR to detect E. canis DNA. When available, blood from these dogs was injected into suckling mice, which were then examined for clinical disease and tested for the presence of E. canis antibodies. RESULTS: Of the 316 samples tested seven reacted in the IFA test for E. canis. None of the dogs from which these samples were obtained exhibited clinical signs of acute or chronic ehrlichiosis. The six positive samples available for testing were negative when tested with the nested PCR. Suckling mice inoculated with blood from three of the dogs whose serum was positive by IFA test showed no signs of clinical disease nor did their give positive reactions in the IFA test. CONCLUSIONS: No evidence of E. canis infection was confirmed in any of the dogs examined. Northern Australia would appear to remain free of this obligate parasite.

Animals↗

High-density microarray-mediated gene expression profiling of Escherichia coli.

A nearly complete collection of 4,290 Escherichia coli open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from E. coli cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established. A brief isopropyl-beta-D-thiogalactopyranoside (IPTG) treatment elevated lacZ, lacY, and lacA transcript content about 30-fold; in contrast, most other transcript titers remained unchanged. Distinct RNA expression patterns between E. coli cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media. The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor. This inventory provided a quantitative view of the steady-state level of each mRNA species. Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of E. coli physiology.

Amino Acids↗

RNA expression analysis using an antisense Bacillus subtilis genome array.

We have developed an antisense oligonucleotide microarray for the study of gene expression and regulation in Bacillus subtilis by using Affymetrix technology. Quality control tests of the B. subtilis GeneChip were performed to ascertain the quality of the array. These tests included optimization of the labeling and hybridization conditions, determination of the linear dynamic range of gene expression levels, and assessment of differential gene expression patterns of known vitamin biosynthetic genes. In minimal medium, we detected transcripts for approximately 70% of the known open reading frames (ORFs). In addition, we were able to monitor the transcript level of known biosynthetic genes regulated by riboflavin, biotin, or thiamine. Moreover, novel transcripts were also detected within intergenic regions and on the opposite coding strand of known ORFs. Several of these novel transcripts were subsequently correlated to new coding regions.

Amino Acid Sequence↗

Global impact of sdiA amplification revealed by comprehensive gene expression profiling of Escherichia coli.

In Escherichia coli the amplification of sdiA, a positive activator of ftsQAZ, genes that are essential for septation, results in mitomycin C resistance. To help us understand this resistance phenotype, genes whose expression was altered by increased sdiA dosage were identified using a DNA microarray-based, comprehensive transcript profiling method. The expression of 62 genes was reduced by more than threefold; of these, 41 are involved in motility and chemotaxis. Moreover, the expression of 75 genes, 36 of which had been previously characterized, was elevated at least threefold. As expected, increased sdiA dosage led to significantly elevated sdiA and 'ddlB-ftsQAZ-lpxC operon expression. Transcription of two genes, uvrY and uvrC, located downstream of sdiA and oriented in the same direction, was elevated about 10-fold, although the intervening gene, yecF, of opposite polarity was unaffected by increased sdiA dosage. Three genes (mioC and gidAB) flanking the replication origin, oriC, were transcribed more often when sdiA dosage was high, as were 12 genes within 1 min of a terminus of replication, terB. Transcription of the acrABDEF genes, mapping in three widely spaced loci, was elevated significantly, while several genes involved in DNA repair and replication (e.g., nei, recN, mioC, and mcrC) were moderately elevated in expression. Such global analysis provides a link between septation and the response to DNA-damaging agents.

Bacterial Proteins↗

Linkage between STAT regulation and Epstein-Barr virus gene expression in tumors.

Epstein-Barr virus (EBV) latency gene expression in lymphoblastoid cell lines is regulated by EBNA2. However, the factors regulating viral expression in EBV-associated tumors that do not express EBNA2 are poorly understood. In EBV-associated tumors, EBNA1 and frequently LMP1 are synthesized. We found that an alternative latent membrane protein 1 (LMP1) promoter, L1-TR, located within the terminal repeats is active in both nasopharyngeal carcinoma and Hodgkin's disease tissues. Examination of the L1-TR and the standard ED-L1 LMP1 promoters in electrophoretic mobility shift assays revealed that both promoters contain functional STAT binding sites. Further, both LMP1 promoters responded in reporter assays to activation of JAK-STAT signaling. Cotransfection of JAK1 or v-Src or treatment of cells with the cytokine interleukin-6 upregulated expression from ED-L1 and L1-TR reporter plasmids. Cotransfection of a dominant negative STAT3 beta revealed that STAT3 is likely to be the biologically relevant STAT for EBNA1 Qp and LMP1 L1-TR promoter regulation. In contrast, LMP1 expression from ED-L1 was not abrogated by STAT3 beta, indicating that the two LMP1 promoters are regulated by different STAT family members. Taken together with the previous demonstration of JAK-STAT activation of Qp driven EBNA1 expression, this places two of the EBV genes most commonly expressed in tumors under the control of the same signal transduction pathway. Immunohistochemical analyses of nasopharyngeal carcinoma tumors revealed that STAT3, STAT5, and STAT1 are constitutively activated in these tumors while STAT3 is constitutively activated in the malignant cells of Hodgkin's disease. We hypothesize that chronic or aberrant STAT activation may be both a necessary and predisposing event for EBV-driven tumorigenesis in immunocompetent individuals.

Cell Line↗

Vertebral bone marrow perfusion evaluated with dynamic contrast-enhanced MR imaging: significance of aging and sex.

PURPOSE: To investigate blood perfusion of nonfractured, normal-appearing vertebral bodies with regard to age and sex. MATERIALS AND METHODS: Dynamic magnetic resonance imaging (160 images obtained in 80 seconds) was performed from T10 to L5 in 66 patients. Patients were assigned to three groups: group 1, those 50 years or younger without compression fracture; group 2, those older than 50 years without compression fracture; or group 3, those older than 50 years with compression fracture. Peak enhancement percentage and enhancement slope were determined from the time-intensity curve of normal (nonfractured) vertebral body. Comparisons were made between groups, and the effect of age and sex interaction was analyzed. RESULTS: Higher peak enhancement percentage was demonstrated for group 1 compared with group 2 (58.21 +/- 44.65 [SD] vs 21.88 +/- 14.77, P <.005). Group 1 women revealed a higher enhancement percentage compared with group 1 men (87.17 +/- 54.13 vs 38.16 +/- 21.69, P <.05), which significantly decreased in those older than 50 years (from 87.17 +/- 54.13 to 17.98 +/- 13.80, P <.005). For men, this decrease in those older than 50 years was not as pronounced (from 38.16 +/- 21.69 to 25.38 +/- 15.43, P >.05). Presence of compression fracture at other levels of the spine (group 3) was not associated with a different enhancement percentage for normal vertebrae. CONCLUSION: Rate of vertebral bone marrow perfusion revealed a significant decrease in subjects older than 50 years. Women demonstrated a higher marrow perfusion rate than men younger than 50 years and a more marked decrease than men older than 50 years.

Adult↗

An inexpensive meter to measure differences in electrical resistance in the rat vagina during the ovarian cycle.

The inherent electrical resistance of the rat vaginal wall rises markedly near the beginning of estrus and then falls again to low levels for the remainder of the ovarian cycle. Accordingly, special instruments have been developed to measure such resistances (within seconds) on simply inserting a small probe fitted with a pair of recording electrodes into the vagina (i.e., the MK-10A impedance checker and the EC40 estrus cycle monitor). As described herein, these two instruments are far more convenient for monitoring individual cycles than more laborious methods in which vaginal smears are inspected for changes in numbers of cornified (C), nucleated (N), and leukocytic (L) cells. However, they are also expensive and their use has essentially remained uncited in the literature. Thus we sought to determine whether a simple, inexpensive electrical meter (with resistance-measuring capacity), as commonly used by professional electricians, would serve the same purpose. We chose a standard multifunctional meter (model 22-178, RadioShack) and attached leads to it fabricated from the internal wiring of a shielded audio cable (model 42-2387A, RadioShack), one male terminal of which was used as a vaginal probe. In rats from which vagina smears revealed cell numbers in the order of C > N > L (typical of early estrus) electrical resistances were high, 488 +/- 130 k Omega (18 rats). In rats from which vagina smears revealed all other possible cell distributions, electrical resistances (combined) were much lower (P < 0.05), 124 +/- 23 k Omega (32 rats). Thus readily accessible, inexpensive electrical meters may be useful in assessing the status of estrus in female rats, either to improve reproductive efficiencies and/or for other purposes involving experiments in which such information is desirable.

Animals↗

Duration of neuronal stretch correlates with functional loss.

Postoperative cranial nerve weakness or paralysis is not uncommon in many otolaryngologic surgical procedures. Our study used a rat model to test the hypothesis that the length of time that a nerve is under tension may be an important variable in the amount of postoperative paresis. Forty Sprague-Dawley rats were divided into 4 groups that underwent either a sham operation or a traction injury for 1, 2, or 5 minutes. The traction injury was performed with a vessel loop placed around the sciatic nerve with 50 g of tension. Traction injury for 1 or 2 minutes did not result in any statistical differences in the motor capabilities of the lower limb. However, those animals with a stretch injury for 5 minutes had a significant loss of function (P < 0.01) when compared with all other groups. Histologic examination of nerves harvested on postoperative day 7 showed no evidence of mechanical injury. This study demonstrates that even minimal tension, if maintained for a significant amount of time, may result in postoperative weakness.

Animals↗

Optimal production and in vitro activity of recombinant endostatin from stably transformed Drosophila melanogaster S2 cells.

Recombinant plasmids containing a complementary deoxyribonucleic acid coding mouse endostatin were transfected and stably expressed in Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing recombinant endostatin were isolated after 4 wk of selection with hygromycin B. Recombinant endostatin expressed in the stably transformed S2 cells under the influence of the Drosophila BiP protein signal sequence was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at maximum inhibition for recombinant endostatin was approximately 1.8 microg/ml. The stably transformed S2 cells produced 18 mg recombinant endostatin/L 7 d after induction with 5 microM CdCl2. Sodium butyrate supplementation (2.5 mM) increased recombinant endostatin production by 17%. These findings demonstrate optimal production and in vitro activity of recombinant endostatin from stably transformed D. melanogaster S2 cells.

Angiogenesis Inhibitors↗