Engineering the thermostable D-hydantoinases from two thermophilic Bacilli based on their primary structures.
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Amidohydrolases↗
Biomedical subjects
D C Lee
Publications and source records attributed to D C Lee.
A cultivation strategy of recombinant Escherichia coli for mass production of thermostable D-hydantoinase.
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Amidohydrolases↗
An essential role for ectodomain shedding in mammalian development.
The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.
ADAM Proteins↗
Secondary structure analysis of the putative membrane-associated domains of the inward rectifier K+ channel ROMK1.
The inward rectifier K+ channels contain two putative membrane-spanning domains per subunit (M1, M2) and a 'pore' (P) region, which is similar to the H5 domain of voltage-gated K+ channels. Here we have used Fourier transform infrared (FTIR) and CD spectroscopy to analyse the secondary structures of synthetic peptides corresponding to the M1, M2 and P regions of ROMK1 in aqueous solution, in organic solvents and in phospholipid membranes. A previous CD study was unable to provide any structural data on a similar P peptide [Ben-Efraim and Shai (1997) Biophys. J. 72, 85-96]. However, our FTIR and CD spectroscopic analyses indicate that this peptide adopts an alpha-helical structure when reconstituted into dimyristoyl phosphatidylcholine vesicles and lysophosphatidyl choline (LPC) micelles as well as in trifluoroethanol (TFE) solvent. This result is in good agreement with a previous study on a peptide corresponding to the pore domain of a voltage-gated K+ channel [Haris, Ramesh, Sansom, Kerr, Srai and Chapman (1994) Protein Eng. 7, 255-262]. FTIR spectra of the M1 peptide in LPC micelles displayed a strong absorbance characteristic of an intermolecular beta-sheet structure, suggesting aggregation of the M1 peptide. Sucrose gradient centrifugation was used to separate aggregated peptide from peptide incorporated into micelles in an unaggregated manner; subsequent analysis by FTIR suggested that the M1 peptide adopted an alpha-helical structure when incorporated into phospholipid membranes. FTIR and CD spectra of the M2 peptide in phospholipids and high concentrations of TFE suggest that this peptide adopts an alpha-helical structure. The structural data obtained in these experiments have been used to propose a model for the structure of the membrane-associated core (M1-P-M2) of the inward rectifier K+ channel protein.
Amino Acid Sequence↗
Sex difference in immunostaining of RET in the adult mouse kidney.
The c-ret proto-oncogene encodes a receptor tyrosine kinase which is important for the development of the kidney and the enteric nervous system. During nephrogenesis, c-ret is expressed in the ureteric bud epithelium and later in its derivative, the collecting duct. This takes place during 11-17.5 days post-coitum (d.p.c.) in the mouse and our immunohistochemical study showed that the RET protein co-localized with the transcript. At 18.5 d.p.c. the kidney is fully differentiated. At 18.5 d.p.c., 1 week and 10 weeks old, RET was found in the proximal convoluted tubules, which is formed from the condensed mesenchyme. This suggests that c-ret may also play a role in kidney function. For the 10 weeks old kidney, RET immunostaining in male was concentrated on the basolateral side while female had a stronger staining in the whole cell. Furthermore, cytoplasmic staining was observed in male whereas both cytoplasmic and nuclear staining was found in female. c-ret transcript was detected by RT-PCR, and in situ hybridization showed its expression throughout the kidney. The reason for the sex-specific staining and the role of RET in kidney function remain to be determined.
Animals↗
Expression of transforming growth factor alpha and epidermal growth factor receptor in adult polycystic kidney disease.
Adult polycystic kidney disease (APKD) is a common genetic disease with a frequency of 1:1000. Evidence suggests that transforming growth factor alpha (TGF alpha) signaling may contribute to the hyperproliferation of the cystic epithelia in APKD. TGF alpha and epidermal growth factor (EGF) are well known mitogens expressed in the kidney and both exert their biological activities through binding to the same EGF receptor. A transgenic mouse that over-expressed TGF alpha developed renal cysts; raised levels of TGF alpha and EGF receptor mRNA were found in kidneys from two autosomal dominant APKD patients. To study the role of TGF alpha in cyst formation, we analyzed nine anatomically diagnosed adult polycystic kidneys and four normal kidneys using immunohistochemistry. We also traced the possible origins of the cysts by staining with the proximal convoluted tubule (PCT) marker, gp330, and the distal convoluted tubule (DCT) and collecting tubule (CT) marker, peanut agglutinin (PNA). In normal kidneys, TGF alpha protein was concentrated in the DCT and CT and EGF receptor protein in all three tubule types. In the early cysts of APKD, the cystic epithelia showed strong positive staining with TGF alpha, EGF receptor and gp330 but negative with PNA. Strong TGF alpha and EGF receptor staining was also found in the mixture of advanced cysts in the end-stage cystic kidneys although the cysts are likely to be derived from different segment of the renal tubules. This increased TGF alpha and EGF receptor expression in all cases and all types of cysts suggests that autocrine/paracrine stimulation by TGF alpha may be a common mechanism in cyst development in APKD.
Adult↗
Hypothermia from realistic fluid resuscitation in a model of hemorrhagic shock.
STUDY OBJECTIVE: To correlate changes in core body temperature with changes in mean arterial pressure (MAP) and cardiac output (CO) and with the administration of room-temperature intravenous fluids in a clinically relevant large-animal model of uncontrolled hemorrhage. METHODS: Ten swine were subjected to uncontrolled hemorrhage through a flow-monitored shunt placed between the femoral artery and the peritoneal cavity. Animals were randomly assigned to a treatment or a control group. The control group (n=5) received no intravenous fluids. The treatment group (n=5) received 80 mL/kg (3:1 crystalloid/blood loss) ambient-temperature lactated Ringer's solution over a 10-minute resuscitation phase initiated 10 minutes after injury. CO and core body temperature, measured with the use of a pulmonary artery catheter, and MAP were the primary outcomes. We analyzed differences between groups with the use of repeated-measures ANOVA. Change of temperature was analyzed against the change in CO, and against fluid infusion for each interval, by means of regression analysis. RESULTS: The unresuscitated control animals had no change in core temperature despite profound hemorrhagic shock and hypotension. The animals treated with fluids had a mean 2.6 degrees C decrease in core temperature during fluid resuscitation (95% confidence interval [CI], 1.8 to 3.5). A 1.5 degrees C decrease in core temperature (95% CI, .1 to 2.0) persisted at the end of 60 minutes (40 minutes after fluid resuscitation was discontinued). Core temperatures in control animals were 2.8 degrees C lower than those in treated animals after fluid resuscitation (95% CI, .8 to 4.8). Decreases in core temperature correlated with fluid infusion (beta=-35.2 mL/kg x degrees C, R2=.75) and increases in CO (beta=-1.46 L/min x degrees C, R2=.69). CONCLUSION: Ambient-temperature crystalloid resuscitation in a clinically relevant large-animal model of hemorrhagic shock causes small decreases in core body temperature. Resuscitation rather than shock is the main cause of decreased body temperature in this model.
Analysis of Variance↗
Acute gastric perforation in a chloral hydrate overdose.
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Abdomen, Acute↗
Intraoperative flexion against gravity as an indication of ultimate range of motion in individual cases after total knee arthroplasty.
To assess a method of predicting the final postoperative flexion in individual cases after total knee arthroplasty, 364 primary posterior cruciate-retaining total knee arthroplasties were reviewed retrospectively. The knees were subdivided into three preoperative flexion groups--I: poor motion (0 degrees to 85 degrees), II: intermediate motion (90 degrees to 110 degrees), and III: good motion (115 degrees to 140 degrees). There were 302 cases of osteoarthritis and 62 rheumatoid knees (12 juvenile rheumatoid). Correlation was made between preoperative; intraoperative, and postoperative (minimum 2-year follow-up) passive knee flexion for individuals. Intraoperative flexion against gravity was measured after capsular closure by passively flexing the patient's hip 90 degrees and allowing the weight of the lower leg to flex the knee joint. The overall mean value of postoperative flexion for all three groups was similar to preoperative and intraoperative flexion in both osteoarthritis and rheumatoid arthritis. In the poor motion group (I), postoperative flexion (103 degrees) was increased over preoperative flexion (84 degrees) but similar to intraoperative flexion (104 degrees). In the intermediate group (II), postoperative flexion (110 degrees) was similar to both the preoperative flexion (108 degrees) and intraoperative flexion (110 degrees). In the good group (III), postoperative flexion (119 degrees) tended to be less than preoperative flexion (123 degrees) and more than intraoperative flexion (116 degrees), but the differences were not statistically significant. When comparing preoperative and intraoperative flexion to postoperative flexion for individual cases, 55% of knees had postoperative flexion +/-10 degrees of their preoperative value, while 97% of knees had postoperative flexion +/-10 degrees of their intraoperative value. This study indicates that the final postoperative mean flexion for a group of patients with poor preoperative flexion (<85 degrees) and for individual cases (regardless of their preoperative mobility) can best be predicted by intraoperative flexion against gravity rather than by a preoperative value.
Arthritis, Juvenile↗
Role of integrins in mouse eyelid development: studies in normal embryos and embryos in which there is a failure of eyelid fusion.
Eyelid fusion normally occurs between E15.5 and E16.5 of mouse embryonic development and results from the migration of a population of periderm-derived epithelial cells over the corneal surface. Cell migration is known to depend on extracellular matrix receptors of the integrin family and to be regulated by growth factors. We were therefore interested that a failure of eyelid fusion has been reported in mice that are homozygous null for the transforming growth factor alpha (TGF-alpha) gene and in mice (invalpha5beta1) in which a transgenic alpha5beta1 integrin under the control of the involucrin promoter is misexpressed in differentiating keratinocytes. We examined expression of the alpha2beta1, alpha3beta1, alpha5beta1 and alpha6beta4 integrins during eyelid fusion in wild-type embryos and found selective upregulation of the alpha5beta1 integrin and its ligand, fibronectin, in the migrating eyelid tip cells. In TGF-alpha null embryos, the failure of eyelid fusion was correlated with a failure to upregulate the alpha5beta1 integrin and fibronectin in the tip cells. Using beta-galactosidase as a reporter gene in transgenic mice, we observed specific activity of the involucrin promoter in the eyelid tip cells. In invalpha5beta1 mice the transgenic human integrin was overexpressed not only in the tip cells but throughout the eyelid epidermis. In contrast, the endogenous, murine, alpha5beta1 integrin was only weakly expressed in the tip cells. We speculate that selective and coordinated expression of the alpha5beta1 integrin and fibronectin in eyelid tip cells is required for eyelid fusion and may be under the control of growth factors that include TGF-alpha.
Animals↗
The use of pamidronate for hypercalcemia secondary to acute vitamin D intoxication.
INTRODUCTION: Pamidronate disodium, a bone resorption inhibitor through osteoclast mediation, has been used to treat chronic hypercalcemia secondary to malignancy and chronic renal failure. We report the use of pamidronate for acute, severe hypercalcemia secondary to iatrogenic vitamin D poisoning. CASE REPORT: A 77-year-old female nursing home resident was inadvertently administered 50,000 units of oral vitamin D daily for 6 days. The patient presented with lethargy, abdominal pain, and vomiting. The patient's initial serum calcium concentration was 5.25 mmol/L (21 mg/dL). The patient was initially treated with hydration and furosemide but developed congestive heart failure. Pamidronate was used and calcium concentrations normalized by 24 hours after treatment. CONCLUSION: We report a case of the use of pamidronate for significant hypercalcemia secondary to acute vitamin D poisoning. Although evidence of congestive heart failure was evident, dialysis was avoided without significant sequelae. Pamidronate therapy should be considered in patients with hypercalcemia secondary to acute vitamin D poisoning.
Aged↗
Dynamic expression and activation of ERBB receptors in the developing mouse mammary gland.
The complex system of ERBB receptors and ligands is implicated in growth and differentiation of the mammary gland. However, it has not been comprehensively examined in this dynamic tissue. Combined RNA and protein analyses of glands in different stages from virgin to involution revealed differential expression of the four ERBB receptors, as well as distinctive patterns of ERBB ligand expression that suggested specialized function. ERBB localization was linked to mammary gland function. Thus, in the virgin gland, ERBB1 and ERBB2 were colocalized to all major cell types during ductal morphogenesis but differentially localized in the mature gland. All four ERBB receptors were restricted to epithelia in the differentiated gland. Analyses of ERBB tyrosine phosphorylation provided strong evidence of interaction between the four receptors in this physiological context. Thus, exogenous EGF induced stage-dependent transphosphorylation of ERBB2-4 as well as ERBB1, whereas endogenous phosphorylation of all four receptors peaked in late pregnancy and lactation.
Animals↗
Transcription factor AP2 is required for expression of the rat transforming growth factor-alpha gene.
DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.
Animals↗
Prenatal ontogeny of the epidermal growth factor receptor and its ligand, transforming growth factor alpha, in the rat brain.
Transforming growth factor alpha (TGF alpha) interacts with the epidermal growth factor receptor (EGF-R) to produce its biological effects. TGF alpha induces the proliferation and differentiation of central nervous system (CNS) astrocytes and pluripotent stem cells, as well as the survival and differentiation of postmitotic CNS neurons. Both TGF alpha and EGF-R have been localized to the postnatal CNS. As the majority of CNS neuronal proliferation and migration occurs antenatally, we have examined the ontogeny of TGF alpha and EGF-R in the embryonic rat brain by in situ hybridization. EGF-R mRNA was expressed in the brain as early as embryonic day 11 (E11; the earliest age examined). It was initially detected in the midbrain, with subsequent expression first in multiple germinal zones, followed by expression in numerous cells throughout the brain. In many brain areas, EGF-R mRNA appeared in germinal centers during the later stages of neurogenesis and the early stages of gliogenesis. In the midbrain, the distribution of EGF-R mRNA overlapped extensively with that of tyrosine hydroxylase mRNA, suggesting that fetal dopaminergic neurons express EGF-R. Immunocytochemistry was used to demonstrate the presence of EGF-R-immunoreactive protein in brain areas that expressed EGF-R mRNA on E15 and E20. The expression of TGF alpha in many brain structures preceded that of EGF-R mRNA. TGF alpha mRNA was distributed throughout many non-germinal centers of the brain on E12 and later. Some brain areas, such as the external granule cell layer of the cerebellum, expressed EGF-R, but not TGF alpha mRNA. Northern blot analysis demonstrated that mRNA species for both TGF alpha and EGF-R were similar in embryos and adults. These data indicate that TGF alpha and EGF-R are positioned to have a role in the genesis, differentiation, migration, or survival of numerous cell populations in the embryonic brain.
Animals↗
Primary structure, sequence analysis, and expression of the thermostable D-hydantoinase from Bacillus stearothermophilus SD1.
The gene coding for the thermostable D-hydantoinase from the thermophilic bacterium Bacillus stearothermophilus SD1 was cloned and its nucleotide sequence was completely determined. The D-hydantoinase protein showed considerable amino acid sequence homology (20-28%) with other hydantoinases and functionally related allantoinases and dihydroorotases. Strikingly the sequence of the enzyme from B. stearothermophilus SD1 exhibited greater than 89% identity with hydantoinases from thermophilic bacteria. Despite the extremely high amino acid homology among the hydantoinases from thermophiles, the C-terminal regions of the enzymes were completely different in both sequence and predicted secondary structure, implying that the C-terminal region plays an important role in determining the biochemical properties of the enzymes. Alignment of the sequence of the D-hydantoinase from B. stearothermophilus SD1 with those of other functionally related enzymes revealed four conserved regions, and five histidines and an acidic residue were found to be conserved, suggesting a close evolutionary relationship between all these enzymes.
Amidohydrolases↗
The carboxyl-terminal valine residues of proTGF alpha are required for its efficient maturation and intracellular routing.
Soluble forms of transforming growth factor-alpha (TGF alpha) are derived by proteolytic processing of an integral membrane glycoprotein precursor (pro TGF alpha). Previous studies indicated that phorbol ester-induced cleavage of pro TGF alpha in CHO cells is dependent on the presence of a valine residue located at the carboxyl terminus of the precursor's cytoplasmic domain. We reassessed this requirement with epitope-tagged constructs introduced into transformed rat liver epithelial cells that normally express and process TGF alpha. We found that pro TGF alpha mutants lacking the terminal valine residues showed greatly reduced maturation to the fully glycosylated form. Additionally, they were present at substantially reduced levels on the cell surface and, instead, accumulated in the endoplasmic reticulum. Consistent with these results, enzyme-linked immunosorbent assay (ELISA) and Western blot analyses revealed little or no soluble TGF alpha in medium conditioned by cells expressing the mutant constructs. Finally, a truncated pro TGF alpha mutant lacking most of the cytoplasmic domain but retaining a carboxyl-terminal valine was processed and cleaved in a near-normal manner. These results, some of which were reproduced in CHO cells, indicate that the predominant effect of the carboxyl-terminal valines is to ensure normal maturation and routing of the precursor.
Animals↗
Characterization of the mouse epidermal growth factor promoter and 5'-flanking region. Role for an atypical TATA sequence.
As a step toward delineating mechanisms that regulate its activity, we have characterized the mouse epidermal growth factor (EGF) promoter. Primer extension and S1 nuclease analyses identified prominent (+1/+2) and minor (+28) transcription start sites, with the dominant +1/+2 site located 33 bases downstream from a TTTAAA sequence. A restriction fragment that spanned these start sites and contained 390 base pairs of 5'-flanking sequence directed transcription from the +1/+2 site in vitro in the presence of HeLa cell nuclear extracts. Additionally, it promoted expression of a coupled luciferase reporter gene in transfected cell lines. The inclusion of additional 5'-flanking sequence either stimulated or inhibited luciferase expression depending on the cell line. Approximately 2 kilobases of EGF 5'-flanking sequence was determined and found to contain several motifs with partial homology to steroid hormone response elements. Despite this fact and evidence that EGF expression might be regulated by androgens in vivo, EGF promoter-luciferase constructs were not steroid-responsive in cells cotransfected with steroid receptor expression vectors. An oligonucleotide containing the aforementioned TTTAAA sequence specifically bound TATA-binding protein and TFIIA in gel shift assays, and an EGF promoter-luciferase construct in which the core TA dinucleotide was mutated to CG was not active in transfected cells. These data suggest that the TTTAAA sequence functions as an atypical TATA box.
Androgens↗
Cloning and overexpression of thermostable D-hydantoinase from thermophile in E. coli and its application to the synthesis of optically active D-amino acids.
We cloned the thermostable D-hydantoinase gene from B. stearothermophilus SD-1 into E. coli. The cloned gene was constitutively expressed by its own promoter, and the enzyme was produced in its soluble form. The specific activity of the recombinant E. coli was 30 times higher than that of B. stearothermophilus SD-1. The cultivation conditions were investigated for the overproduction of the enzyme, and the temperature was found to affect the plasmid content and the expression level of the enzyme. Recombinant E. coli was cultivated in 30-L batch fermentation, the cell concentration reached 25 g-DCW/L, and the specific activity was about 20,000 units/g-DCW. D-Hydantoinase produced from the recombinant E. coli could be successfully applied to the synthesis of N-carbamoyl-D-amino acid from the 5-monosubstituted hydantoin derivative.
Amidohydrolases↗