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B Y Lee

Publications and source records attributed to B Y Lee.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification of fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iron by combined two-dimensional gel electrophoresis and mass spectrometry.

Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 microM) and high-iron (70 microM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance.

Aconitate Hydratase↗
PubMed1999

T-cell epitope mapping of the three most abundant extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs.

The three most abundant extracellular proteins of Mycobacterium tuberculosis, the 30-, 32-, and 16-kDa major extracellular proteins, are particularly promising vaccine candidates. We have mapped T-cell epitopes of these three proteins in outbred guinea pigs by immunizing the animals with each protein and assaying splenic lymphocyte proliferation against a series of overlapping synthetic peptides covering the entire length of the mature proteins. The 30-kDa protein contained nine immunodominant epitopes, the 32-kDa protein contained two immunodominant epitopes, and the 16-kDa protein contained a highly immunodominant region at its N terminus. The immunodominant epitopes of the 30- and 32-kDa proteins in outbred guinea pigs were frequently identified in healthy purified-protein-derivative-positive or BCG-vaccinated individuals in previous studies. The immunodominant epitopes of these major extracellular proteins have potential utility in an epitope-based vaccine against tuberculosis.

Amino Acid Sequence↗
PubMed1998

California ground squirrel body temperature regulation patterns measured in the laboratory and in the natural environment.

Body temperature (Tb) was measured by telemetry in both laboratory maintained and natural environment California ground squirrels, Spermophilus beecheyi. Laboratory animals had a mean diurnal Tb of 37.5 degrees C under conditions of LD 14:10, 20 degrees C and 36.5 degrees C under conditions of LD 10:14, 20 degrees C (P < 0.01). Nocturnal mean Tbs were 37.1 and 35.2 degrees C, respectively (P < 0.05). Mean diurnal Tbs for each animal in the natural environment ranged from 39.3 to 40.1 degrees C (mean = 39.6 degrees C) during both study seasons which included the hot season months of March through August and the cool season months of December through February. Natural environment hot season mean Tb was not significantly different from cool season mean Tb but both mean Tbs were significantly different from the diurnal mean Tbs measured in the laboratory (P < 0.05). California ground squirrels exhibit an open-field stress induced hyperthermia in the laboratory which can be extended for periods up to 6 h. The hyperthermic response is blocked by L-propranolol at a dosage of 15 mg kg-1. Laboratory animals do not habituate to repeated open-field exposures over a five consecutive day period. It is suggested that stress hyperthermia might be a normal component of thermoregulation in some free-living ground squirrels because of the openness of the habitat in which they exist.

Animals↗
PubMed1998

The yiaE gene, located at 80.1 minutes on the Escherichia coli chromosome, encodes a 2-ketoaldonate reductase.

An open reading frame located in the bisC-cspA intergenic region, or at 80.1 min on the Escherichia coli chromosome, encodes a hypothetical 2-hydroxyacid dehydrogenase, which was identified as a result of the E. coli Genome Sequencing Project. We report here that the product of the gene (yiaE) is a 2-ketoaldonate reductase (2KR). The gene was cloned and expressed with a C-terminal His tag in E. coli, and the protein was purified by metal-chelate affinity chromatography. The determination of the NH2-terminal amino acid sequence of the protein defined the translational start site of this gene. The enzyme was found to be a 2KR catalyzing the reduction of 2, 5-diketo-D-gluconate to 5-keto-D-gluconate, 2-keto-D-gluconate (2KDG) to D-gluconate, 2-keto-L-gulonate to L-idonate. The reductase was optimally active at pH 7.5, with NADPH as a preferred electron donor. The deduced amino acid sequence showed 69.4% identity with that of 2KR from Erwinia herbicola. Disruption of this gene on the chromosome resulted in the loss of 2KR activity in E. coli. E. coli W3110 was found to grow on 2KDG, whereas the mutant deficient in 2KR activity was unable to grow on 2KDG as the carbon source, suggesting that 2KR is responsible for the catabolism of 2KDG in E. coli and the diminishment of produced 2KDG from D-gluconate in the cultivation of E. coli harboring a cloned gluconate dehydrogenase gene.

Alcohol Oxidoreductases↗
PubMed1998

Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum.

Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of p10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected Trichoplusia ni larvae by the same period.

Amino Acid Sequence↗
PubMed1997

Nutritional disorders in a concentration camp.

OBJECTIVE: Observations on nutritional disorders were made by a physician inmate in a concentration camp before and during the Great Starvation in China. METHODS/RESULTS: Based on therapeutic response, many unique abnormalities should be considered as a part of clinical picture of malnutrition, such as mucocutaneous pigmentation, nail layering phenomena and intranail hemorrhage, palmar/plantar fissures, vegetative system crisis, a avitaminostic fevers, multiple premature beats, and enlargement of cartilage, lymph nodes, and submandibular glands. DISCUSSION: Thiamin deficiency should be also considered as one, if not the only, etiologic factor of several common disorders, including submandibular gland cyst, Baker's cyst, stenosing tenosynovitis, direct inguinal hernia, among others.

Adult↗
PubMed1997

Streaming potentials in gap osteotomy callus and adjacent cortex. A pilot study.

This study documented streaming potentials generated in vivo by maturing osteotomy calluses in 10 canine tibiae. Gap osteotomies were allowed to heal for 6 or 12 weeks and were stabilized by an external fixator. Then, with the dogs under anesthesia, electrical measurements were made from 3 silver-silver chloride electrodes placed surgically in direct contact with the callus, with adjacent cortical bone, and with the medullary canal (reference electrode). Streaming potentials were recorded during step loading and sinusoidal bending (0.1-30 Hertz) as the tibia was deformed by 2 threaded pins coupled to a servohydraulic device. Streaming potentials were generated at callus and adjacent cortical sites, but the magnitude was greater on the immature, flexible callus, where bending strain was concentrated; as the callus became increasingly rigid, strain and streaming potential magnitude were distributed more evenly over the callus and adjacent cortical fragments. When normalized to surface strain, mean streaming potential per strain was less dependent on the microscopic structure, although on individual specimens streaming potential per strain at callus and adjacent cortical bone sites tended to increase with decreasing porosity. Despite a wide variation in data in this pilot series, these observations are consistent with the natural history of callus maturation: the maximum magnitude of streaming potentials in callus appears to decrease as the strain gradient across the site decreases, whereas streaming potentials normalized to strain increase as bone matures and becomes more dense.

Animals↗
PubMed1997

High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets.

Mycobacterium tuberculosis, the primary etiologic agent of tuberculosis, is the world's leading cause of death from a single infectious agent, and new vaccines and drugs to combat it are urgently needed. The major extracellular proteins of M. tuberculosis, which are released into its phagosome in macrophages, its host cells in humans, are leading candidates for a vaccine and prime targets for new drugs. However, the development of these biologicals has been hampered by the unavailability of large quantities of recombinant extracellular proteins identical to their native counterparts. In this report, we describe the heterologous expression and secretion of four major M. tuberculosis extracellular proteins (the 30-, 32, 16-, and 23.5-kDa proteins--the first, second, third, and eighth most abundant, respectively) in rapidly growing, nonpathogenic mycobacterial species. Multiple attempts to obtain secretion of the proteins by using Escherichia coli- and Bacillus subtilis-based expression systems were unsuccessful, suggesting that high-level expression and secretion of these Mycobacterium-specific proteins require a mycobacterial host. All four recombinant proteins were stably expressed from the cloned genes' own promoters at yields that were 5- to 10-fold higher than those observed for the native proteins. The four proteins were purified to apparent homogeneity from culture filtrates by ammonium sulfate precipitation and ion-exchange and molecular sieve chromatography. The recombinant proteins were indistinguishable from their native counterparts by multiple criteria. First, N-terminal amino acid sequence determination demonstrated that processing of the leader peptides was highly accurate. Second, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed identical migration patterns. Third, mass spectrometry analysis confirmed that differences in mass were < or = 5 Da. A homolog of the M. tuberculosis 30-kDa protein was identified in M. smegmatis by means of DNA analyses and immunoscreening. This is the first time that secretion of recombinant M. tuberculosis extracellular proteins in their native form has been achieved. This study opens the door to mass production of correctly processed and secreted extracellular proteins of M. tuberculosis in a heterologous host and allows ready evaluation of their biologic and immunologic function.

Amino Acid Sequence↗
PubMed1997

Noninvasive quantification of muscle oxygen in subjects with and without claudication.

The disabling pain of intermittent claudication (IC) arises from oxygen deprivation in the lower limbs during walking. Measurement of the oxygen deficiency within the limb tissue now appears possible with recently expanded understanding of the photon transport through tissue for photons in the visible and near infrared range. Noninvasive measurement consists of preferentially measuring photons that have traveled more deeply into limb tissues and that, therefore, may reach locations of ischemic tissue. Oxygen measurements appear to be possible up to a depth approaching 1.5 cm beneath the surface of the skin. The present study reports on data acquired from the limbs of 11 subjects with IC and 12 subjects without IC. The subjects with IC are patients with clinical findings of claudication based upon segmental Doppler pressure profiles and subjective reports by the patient of pain during exercise. The subjects without IC are individuals with no prior history of ischemic vascular disease. The results consist of photon reflectance measurements at red and infrared wavelengths (approximately 660 nm and 880 nm respectively) taken before, during, and after exercise. Infrared reflectance indices are plotted as well as oxygenation indices generated from combining red and infrared reflectances. A compilation of exercise data shows responses that are generally consistent with the expected physiological responses to mild exercise in subjects with and without IC. We anticipate that the findings of this study may lead to an objective noninvasive testing procedure for measuring the ischemic and exercise-induced changes in muscle oxygenation in the presence of claudication. If the testing of ischemic hypoxia continues to show consistency and accuracy in determining the disability of the subjects with IC, future studies can more effectively test modes of conservative management, such as cessation of smoking, alternative exercise regimens, weight loss, and alternative pharmacological agents.

Exercise↗
PubMed1996

Formation of abdominal adhesions is inhibited by antibodies to transforming growth factor-beta1.

Transforming growth factor-beta (TGF-beta) is an important factor in regulating the inflammatory response and the production of extracellular matrix by fibroblasts. These two processes are linked in the formation of fibrous adhesions after abdominal surgery. When the mesothelium is injured a fibrin strand is produced which is populated first by inflammatory cells then by fibroblasts which secrete extracellular matrix forming a permanent adhesion. TGF-beta promotes both chemotaxis of monocytes and the production of extracellular matrix by fibroblasts. We have used a model of abdominal adhesions in rats in which a circle of peritoneum is dissected and then sutured into place again. After 2 weeks the rats are euthanized and the adhesions are scored. Six groups of 10 rats each underwent this surgery. Group I served as the operative control. Group II was treated with saline which was injected immediately after surgery and on Days 1 and 2 after surgery (vehicle control). Using the same protocol with saline as vehicle, the other four groups of rats were treated with nonspecific IgG (150 microgram per day), anti-TGF-beta (panspecific, 167 microgram per day), anti-TGF-beta1 (67 microgram per day), or anti-TGF-beta2 (50 microgram per day). The rats injected with anti-TGF-beta1 had significantly lower adhesion scores (P < 0.05) than the controls. Rats injected with anti-TGF-beta2 or anti-TGF-beta (panspecific) did not differ significantly from the control saline-injected rats. The results indicate that specifically reducing levels of TGF-beta1 alone can be effective in preventing abdominal adhesions.

Animals↗
PubMed1996

The correlation of ankle oscillometric blood pressures and segmental pulse volumes to Doppler systolic pressures in arterial occlusive disease.

PURPOSE: This study was designed to evaluate the accuracy and failure rates of automatically collected oscillometric ankle systolic pressures (Psys) and pulse volumes (Pvol) using a new algorithm as compared with Psys obtained by standard manual Doppler-and-cuff technique. METHODS: One hundred ten consecutive patients at a vascular laboratory had brachial and ankle Psys measured with the two methods. Pvol at or near the mean arterial pressure was also obtained automatically by the oscillometric device. RESULTS: Both methods showed a 6.6% failure rate when measuring Psys at the ankle. Oscillometric Psys measurement was possible when Doppler Psys failed as a result of nonoccluding arteries. No difference was found between the two methods in occluding limbs with ankle-brachial indexes of 1.30 or more. Sequential brachial Psys values had a mean difference (Doppler-oscillometric) or 2 +/- 10.9 mm Hg and a correlation coefficient (r) of 0.92. Measurements at the ankle had a mean difference of -8.4 +/- 16.8 mm Hg and r = 0.90. These differences were not statistically significant. Mean arterial pressure Pvol recorded at the ankle also correlated with ankle Doppler Psys (r = 0.71) and showed a 1.9% failure rate. CONCLUSION: Both automatic oscillometric plethysmographic Psys and Pvol at the ankle are shown to correlate well with Doppler-and-cuff Psys in patients with vascular disease. Oscillometric measurements can replace Doppler measurements in most clinical situations.

Adult↗
PubMed1996

Surgery of the sympathetic nervous system.

This article reviews the innervation of the arterial system of the lower extremity, lumbar sympathectomy in vascular surgery, lumbar sympathectomy for digital gangrene and in the prevention of major amputation of the lower extremity and substance P's role in neurogenic inflammatory modulation. Long-term results of lumbar sympathectomy and direct arterial bypass surgery have also been reviewed. In addition to the pilomotor, sudomotor and vasomotor actions of the sympathetic nervous system via its neurotransmitters, the molecular basis of the chronic neurogenic inflammatory reaction have been addressed with special attention to the discovery of substance P in the lumbar sympathetic chain and ganglia of human beings.

Amputation, Surgical↗
PubMed1996

Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton major extracellular protein of Mycobacterium tuberculosis.

The 30/32-kDa complex of major secretory proteins are among the most important and intensively studied proteins of Mycobacterium tuberculosis. The proteins have been demonstrated to be immunoprotective and to play a central role in the physiology of the mycobacterium. In this study, we present a series of novel insights into this key protein complex arising out of a combination of genetic, biochemical, and immunocytochemical analyses. Our genetic analyses (i) indicate that the genes are arranged as separate transcription units, (ii) demonstrate that the mature 30-kDa protein of M. tuberculosis differs from the corresponding 30-kDa proteins of two strains of Mycobacterium bovis BCG by only 1 and 5 amino acids, (iii) suggest that expression of the proteins is regulated at the transcriptional level, and (iv) map the transcriptional start site of the 30-kDa protein gene. Our biochemical analyses provide evidence that (i) the 30-kDa protein and the two 32-kDa proteins (i.e., 32A and 32B) are secreted at a ratio of approximately 3:2:1, respectively, (ii) the proteins exist as monomers, (iii) the proteins are not posttranslationally modified by the addition of carbohydrates and lipids, (iv) the 30-kDa and 32A proteins contain one disulfide bridge, and (v) high-level expression and leader peptide processing are achievable in Escherichia coli. Our immunocytochemical analyses demonstrate that the 30/32-kDa complex is expressed in human monocytes and that the proteins are localized to the phagosomal space and the mycobacterial cell wall. These analyses fill important gaps in our knowledge of this critical protein complex of M. tuberculosis and, at the same time, raise new and fundamental questions regarding regulatory mechanisms that control coordinate expression of the proteins at a fixed ratio.

Amino Acid Sequence↗
PubMed1995

Gonadal steroids modulate interleukin-1 receptor antagonist mRNA expression in cultured human monocytes.

This study investigated whether gonadal steroids modulate the expression of the cytokine Interleukin-1 receptor antagonist in monocytes. Human male peripheral monocytes were isolated and cultured in serum free media with serially diluted concentrations of estradiol and progesterone. mRNA expressions with increasing steroid concentrations were compared by reverse transcription-polymerase chain reaction for intracellular and secretory interleukin-1 receptor antagonist specific primers and glyceraldehyde 3-phosphate dehydrogenase primers. Monocyte expression of secretory Interleukin-1 receptor antagonist mRNA was significantly elevated in the presence of normal physiological levels of estradiol (10(-11) M) and progesterone (10(-8) M), while expression was suppressed by higher concentrations of steroids. Intracellular receptor antagonist was also detected. This study is the first to describe the dose related response of cytokine interleukin-1 receptor antagonist to gonadal steroids.

Base Sequence↗
PubMed1995

Autonomic dysreflexia revisited.

Autonomic dysreflexia (AD) is a clinical phenomenon that affects patients with spinal cord injury (SCI) above the major sympathetic outflow tract. The lesion is most often at or above the T-6 level. Any noxious stimuli below this level initiate reflex sympathetic activity resulting in life threatening hypertension uncontrollable by the feedback parasympathetic activity. The episodes of hypertension generally persist until the offending stimulus is removed. Absence of higher control over reflex sympathetic activity due to transection of the cord is an anatomical explanation of the phenomenon. Current evidence suggests additional factors such as supersensitivity and possibly increased numbers of spinal alpha adrenoreceptors and peripheral microvascular adrenoreceptors as well as accumulation of substance P below the lesion. It has been suggested that substance P acts as a modulator, initiating the sympathetic event to produce a strong, slow and prolonged excitatory action. Autonomic dysreflexia is further accentuated by the absence of gamma amino benzoic acid (GABA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT) below the lesion. GABA is an inhibitory neurotransmitter. It has been suggested that either NE or 5-HT may also act as an inhibitory neurotransmitter. Resetting of the baroreceptors at a lower level also plays an important role. The anatomical transection at or above T-6 then helps in maintaining and accentuating the biochemical changes that develop in patients with high spinal cord lesions. The current article reviews the pathophysiology and management of this potentially life threatening, yet easily treatable, phenomenon.

Autonomic Nervous System Diseases↗
PubMed1995

Pulmonary blastoma presenting as a solitary lip metastasis: case report and review of the literature.

Pulmonary blastoma (PB) is an uncommon primary lung malignancy. This neoplasm was first described by Barrett and Barnard in 1945. The tumor is composed of immature epithelial and mesenchymal tissues which may recapitulate early embryological lung development. Under the microscope, the globular component resembles immature bronchus and connective tissue as seen in embryonic lung. More than one hundred cases have been reported in the literature. PB is more frequent in older people and in males and tends to affect blacks at younger ages. Symptomatology varies from asymptomatic to symptoms of a non-specific pulmonary disease. Cough, hemoptysis, dyspnea, chest pain, respiratory distress, fever, anorexia and weight loss are the most common presenting features. The most common roentgenologic pattern is a well-demarcated peripheral lesion, encapsulated by compression or atelectatic lung tissue, although in some cases there is a tendency to lobulation and cavitation. The size of the mass varies from a small peripheral nodule to a mass occupying the entire lobe or hemithorax. The treatment of choice has been surgical excision, radiation and, in selected cases, a combination of chemotherapy with radiation. The prognosis of this malignancy is poor; overall five-year survival is approximately 16 percent. No correlation has been established between histopathologic criteria and survival. The factors that indicate poor prognosis are tumor recurrence, metastasis at initial presentation, tumor size over 5 cm and lymph node metastasis. Liver, central nervous system and bones are the most frequent location of distant metastases. A rare case is presented of a pulmonary blastoma with an upper lip metastasis occurring in a paraplegic male. Diagnosis was confirmed by autopsy findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1995

Role of embryonic factors in human implantation.

Implantation biology is now at a stage where experimental science will be very productive in answering basic questions about the ability of an embryo to implant. The advancement of our knowledge of cytokines and growth factors has been critically important in fuelling the recent new understanding of embryo implantation. Specifically, our increased knowledge of the interleukin (IL)-1 system, as well as leukaemia-inhibiting factor (LIF), epidermal growth factor and colony-stimulating factor-1, and the availability of recombinant protein, specific antibodies and knockout mice, have led to a more detailed outline of implantation events. LIF and IL-1 are the two systems where recent advances have suggested their importance in implantation events. Recently, LIF has been shown in mice to be an endometrial requirement for implantation and embryo development. Although LIF is a pleiotropic molecule, with many interactions in multiple body tissues, in the uterus, concentrations are elevated on day 4 of pregnancy. Experiments with knockout mice have shown the requirement for endometrial LIF for successful implantation. The IL-1 system, consisting of two agonists (IL-1 alpha and IL-1 beta), two receptors (IL-1R types I and II) and the homologous IL-1 receptor antagonist (ra), has also been studied. Knowledge that the embryo secretes IL-1 suggested the interaction between embryonic IL-1 and endometrial receptor, which has been shown to occur. IL-1R type I is plentiful on endometrial epithelial cells and appears to interact with embryonically secreted IL-1 beta to favour implantation. Such implantation events in vivo in mice are blocked by the introduction of large quantities of IL-1ra, consistent with the hypothesis that appropriate interactions between agonist and receptor at the level of the endometrial surface are a requisite for successful implantation. As more specific information on each cytokine or growth factor system comes to light, more complete information on the multiple molecular steps of implantation will become apparent. However, it is clear that no single cytokine or growth factor will be able to explain the complicated events of embryo implantation. Such an important necessary phenomenon has multiple redundancies. The interactions between cytokines and growth factors are becoming increasingly apparent and will need more experimental evidence before a full understanding of implantation is available.

Embryo Implantation↗