Biomedical subjects

Z Sun

Publications and source records attributed to Z Sun.

At least 19 recordsLinked to original sources

PubMed1999

Protein structure prediction in a 210-type lattice model: parameter optimization in the genetic algorithm using orthogonal array.

We have applied the orthogonal array method to optimize the parameters in the genetic algorithm of the protein folding problem. Our study employed a 210-type lattice model to describe proteins, where the orientation of a residue relative to its neighboring residue is described by two angles. The statistical analysis and graphic representation show that the two angles characterize protein conformations effectively. Our energy function includes a repulsive energy, an energy for the secondary structure preference, and a pairwise contact potential. We used orthogonal array to optimize the parameters of the population, mating factor, mutation factor, and selection factor in the genetic algorithm. By designing an orthogonal set of trials with representative combinations of these parameters, we efficiently determined the optimal set of parameters through a hierarchical search. The optimal parameters were obtained from the protein crambin and applied to the structure prediction of cytochrome B562. The results indicate that the genetic algorithm with the optimal parameters reduces the computing time to reach a converged energy compared to nonoptimal parameters. It also has less chance to be trapped in a local energy minimum, and predicts a protein structure which is closer to the experimental one. Our method may also be applicable to many other optimization problems in computational biology.

Algorithms↗

PubMed1999

Reduced pyrazinamidase activity and the natural resistance of Mycobacterium kansasii to the antituberculosis drug pyrazinamide.

Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug that requires conversion to the bactericidal compound pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) activity of nicotinamidase to show activity against Mycobacterium tuberculosis. Mutations leading to a loss of PZase activity cause PZA resistance in M. tuberculosis. M. kansasii is naturally resistant to PZA and has reduced PZase activity along with an apparently detectable nicotinamidase activity. The role of the reduction in PZase activity in the natural PZA resistance of M. kansasii is unknown. The MICs of PZA and POA for M. kansasii were determined to be 500 and 125 micrograms/ml, respectively. Using [14C]PZA and [14C]nicotinamide, we found that M. kansasii had about 5-fold-less PZase activity and about 25-fold-less nicotinamidase activity than M. tuberculosis. The M. kansasii pncA gene was cloned on a 1.8-kb BamHI DNA fragment, using M. avium pncA probe. Sequence analysis showed that the M. kansasii pncA gene encoded a protein with homology to its counterparts from M. tuberculosis (69.9%), M. avium (65.6%), and Escherichia coli (28.5%). Transformation of naturally PZA-resistant M. bovis BCG with M. kansasii pncA conferred partial PZA susceptibility. Transformation of M. kansasii with M. avium pncA caused functional expression of PZase and high-level susceptibility to PZA, indicating that the natural PZA resistance in M. kansasii results from a reduced PZase activity. Like M. tuberculosis, M. kansasii accumulated POA in the cells at an acidic pH; however, due to its highly active POA efflux pump, the naturally PZA-resistant species M. smegmatis did not. These findings suggest the existence of a weak POA efflux mechanism in M. kansasii.

Amidohydrolases↗

PubMed1998

Differential expression of two isopentenyl pyrophosphate isomerases and enhanced carotenoid accumulation in a unicellular chlorophyte.

The enzyme isopentenyl pyrophosphate (IPP) isomerase catalyzes the reversible isomerization of IPP to produce dimethylallyl pyrophosphate, the initial substrate leading to the biosynthesis of carotenoids and many other long-chain isoprenoids. Expression of IPP isomerase, and of two enzymes specific to the carotenoid pathway (lycopene beta-cyclase and beta-carotene-C-4-oxygenase), was followed in the green unicellular alga Haematococcus pluvialis after exposure to high illumination. This alga uniquely accumulates carotenoids in the cytoplasm and in late developmental stages turns deep-red in color because of accumulation of ketocarotenoids in the cytosol. The carotenoid/chlorophyll ratio increased 3-fold in wild type and 6-fold in a precocious carotenoid-accumulating mutant (Car-3) within 24 h after increasing the illumination from 20 to 150 micromol photon m-2.s-1. Two cDNAs encoding IPP isomerase in Haematococcus, ipiHp1 and ipiHp2, were identified. Although otherwise highly similar (95% identity overall), the predicted sequence of ipiHp1 contained a 12-aa region not found in that of ipiHp2. This was reflected by a size difference between two polypeptides of 34 and 32.5 kDa, both of which reacted with an antibody to the product of ipiHp1. We suggest that the 32.5-kDa form is involved with the carotenoid accumulation in the cytoplasm, since the 32.5-kDa polypeptide was preferentially up-regulated by high light preceding the carotenoid increase and only this form was detected in red cysts.

Amino Acid Sequence↗

PubMed1998

Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint.

The Rad53 protein kinase of Saccharomyces cerevisiae is required for checkpoints that prevent cell division in cells with damaged or incompletely replicated DNA. The Rad9 protein was phosphorylated in response to DNA damage, and phosphorylated Rad9 interacted with the COOH-terminal forkhead homology-associated (FHA) domain of Rad53. Inactivation of this domain abolished DNA damage-dependent Rad53 phosphorylation, G2/M cell cycle phase arrest, and increase of RNR3 transcription but did not affect replication inhibition-dependent Rad53 phosphorylation. Thus, Rad53 integrates DNA damage signals by coupling with phosphorylated Rad9. The hitherto uncharacterized FHA domain appears to be a modular protein-binding domain.

Amino Acid Sequence↗

PubMed1998

Analysis of the forces which stabilize the active conformation of urokinase-type plasminogen activator.

It was recently proposed that hydrophobic interactions control the active conformation of serine proteases in the trypsin family (Hedstrom et al. (1996) Biochemistry 35, 4515-23) rather than a charge interaction with Asp next to the active site Ser, as formerly believed. In the present study, certain site-directed mutants of the serine protease zymogen pro-urokinase (pro-UK) and its two-chain enzymatic derivative urokinase (UK) were characterized. The results provide information on the structure-function of the catalytic domain of pro-UK/UK, which is relevant to this controversy. Mutations at Asp355(c194), which eliminated its charge, induced a 6250-fold reduction in the catalytic activity of UK. By contrast, reducing the hydrophobicity at the neoterminal Ile159(c16) of UK had relatively little effect. However, when both the hydrophobicity and the size of the side chain were reduced by a glycine substitution at this position, a major reduction (9090-fold) in the catalytic efficiency of UK occurred. This effect was related to the smaller side chain increasing the cavity and the flexibility of the N-terminus and thereby interfering with its charge interaction with Asp355(c194). A similar mechanism, rather than a change in hydrophobicity, is believed also to explain the reduction in the stabilization energy of the activation domain observed in a trypsin mutant by Hedstrom et al. (1996). Although hydrophobic interaction facilitated the charge interaction with Asp355(c194), the latter was the primary force which stabilized the active conformation of UK. The charge interaction with Asp355(c194) was also found to be the principal determinant of the intrinsic catalytic activity of single-chain pro-UK. Additionally, the findings confirmed that the KM of pro-UK for its natural substrate was significantly lower than that of UK. Since this same phenomenon was also seen with each of the mutants, the substrate binding pocket of these single-chain zymogens was better formed than that of their two-chain, enzymatic derivatives.

Aspartic Acid↗

PubMed1998

Two promoters direct expression of the murine Spi-B gene, an Ets family transcription factor.

Spi-B and PU.1 (Spi-1) comprise the most divergent subfamily of the Ets transcription factor family. Spi-B and PU.1 bind to similar DNA sequences, and can activate the same B-cell and myeloid promoters in vitro. However, PU.1 knockout mice demonstrate defective hematopoietic development of multiple hematopoietic lineages, indicating that Spi-B was not able to compensate for loss of PU.1. One explanation for these results is that, in contrast to PU.1, which is expressed in myeloid and B-cell lines, Spi-B expression is restricted to B-cells. In order to begin to understand the control of regulation of the Spi-B gene, murine Spi-B cDNA and genomic clones were isolated. The exon/intron organization and transcriptional start sites were determined; two major transcriptional start sites were detected. The two Spi-B promoters were isolated and characterized, and displayed differential activity in B-cell lines matching that of the endogenous gene. Further study of the two Spi-B promoters will provide insight into the molecular events regulating the tissue-specific and developmental stage-specific expression of Spi-B in B-cells.

Amino Acid Sequence↗

PubMed1998

The Hancock II bioprosthesis at 12 years.

BACKGROUND: The Hancock II bioprosthesis has been used for heart valve replacement since 1982 in our institution. We previously reported its clinical performance at 8 years and at 10 years. This is a progress report on its performance at 12 years. METHODS: From 1982 to 1994 the Hancock II bioprosthesis was used for aortic valve replacement (AVR) in 723 patients and for mitral valve replacement (MVR) in 328 patients. The mean age of the patients was 65 years for both groups. Coronary artery disease was present in 42% of patients who had AVR and 45% of patients who had MVR. Patients have been followed up prospectively at annual intervals; the mean follow-up was 68+/-40 months for AVR and 66+/-43 months for MVR; it was 99% complete. RESULTS: There were 36 (5%) operative and 159 late deaths in the AVR group, and 26 (8%) operative and 92 late deaths in the MVR. The actuarial survival at 12 years was 54%+/-4% for AVR and 42%+/-5% for MVR. Age greater than 65 years and coronary artery disease had a profound effect on late survival. At 12 years the freedom from thromboembolism was 86%+/-2% for AVR and 90% +/-2% for MVR; from endocarditis, 95%+/-1% for both groups; from primary tissue failure, 94%+/-2% for AVR and 82%+/-5% for MVR; and from valve reoperation, 89% +/-3% for AVR and 78%+/-5% for MVR. There was no primary tissue failure at 12 years in patients older than 65 years who had AVR. CONCLUSIONS: The clinical performance of the Hancock II has been very satisfactory and this bioprosthesis appears to be more durable than its predecessors.

Adolescent↗

PubMed1998

Long-term results of mitral valve repair for myxomatous disease with and without chordal replacement with expanded polytetrafluoroethylene sutures.

OBJECTIVE: This study was carried out to evaluate the long-term results of mitral valve repair for mitral regurgitation caused by myxomatous disease of the mitral valve and the late effects of chordal replacement with expanded polytetrafluoroethylene sutures in this operation. METHODS: A total of 324 patients with mitral regurgitation caused by myxomatous disease underwent mitral valve repair from 1981 to 1995; the group comprised 241 men and 83 women whose mean age was 58 +/- 14 years. Chordal replacement with expanded polytetrafluoroethylene sutures has been performed in 165 patients since 1985. Most of the patients who had chordal replacement with expanded polytetrafluoroethylene sutures had prolapse of the anterior leaflet or prolapse of both leaflets, whereas most patients who had mitral valve repair without chordal replacement had prolapse of the posterior leaflet. Patients were followed up at annual intervals and had a Doppler echocardiographic study. The follow-up was complete and extended from 6 to 156 months (mean 36 +/- 30 months). RESULTS: Two operative and 21 late deaths occurred (14 cardiac and 7 noncardiac). At 10 years the actuarial survival was 75% +/- 5%, the freedom from stroke was 94% +/- 2%, the freedom from transient ischemic attacks was 92% +/- 4%, the freedom from endocarditis was 99% +/- 1%, the freedom from mitral valve reoperation was 96% +/- 1%, and the freedom from severe mitral regurgitation was 93% +/- 3%. Chordal replacement with expanded polytetrafluoroethylene sutures had no effect on any of these end points. CONCLUSIONS: Mitral valve repair was feasible in most patients with mitral regurgitation caused by myxomatous disease and it was associated with low rates of valve-related complications. Chordal replacement with expanded polytetrafluoroethylene had no adverse effect on the late outcome and was believed to have increased the probability of mitral valve repair.

Adolescent↗

PubMed1998

The combination of photocatalysis and ozonolysis as a new approach for cleaning 2,4-dichlorophenoxyaceticacid polluted water.

Treatment of 2,4-D polluted waters with photocatalysis leads to the buildup of high concentrations of the long living intermediate 2,4-DCP. A new approach using a combination of ozonolysis and photocatalysis gave better degradation results with lower intermediate concentrations. The advantages of photocatalysis giving a constant decline in TOC and of ozonolysis giving no buildup of high intermediate concentrations were combined. Degradation data of 2,4-D for photocatalysis, ozonolysis and the combination of both for different pH ranges are given. Data on the main intermediate 2,4-DCP are given for the three different approaches.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1998

The influence of apoptosis on intestinal barrier integrity in rats.

BACKGROUND: Apoptosis is a critical step responsible for maintaining the cellular balance between proliferation and death and for controlling tumorigenesis. Although an increase in intestinal apoptotic cells has been considered to be associated with the pathogenesis of gastrointestinal injury, little is understood concerning the role of apoptosis in the development of intestinal barrier dysfunction. METHODS: Apoptosis induced by intraperitoneal injection of doxorubicin in rats was evaluated by transmission electron microscopy and the TUNEL histochemistry method. Treatment with deoxy-D-glucose (a glycolytic pathway inhibitor) or cycloheximide (a protein synthesis inhibitor) was performed after doxorubicin challenge. Passage of human serum albumin from blood to the intestinal interstitium and the intestinal lumen or from the intestine to the intestinal interstitium and blood was evaluated by means of albumin clearance. RESULTS: A significant increase in gut water content, albumin flux, and bidirectional clearance of albumin accompanied by apoptotic epithelial cell increase was noted in doxorubicin-challenged rats treated with saline. The increase in endothelial and epithelial permeability and the increase of apoptosis could partly be prevented by treatment with deoxy-D-glucose or cycloheximide. CONCLUSION: Doxorubicin-increased epithelial apoptosis within the intestine occurs simultaneously with increased bidirectional permeability of the intestinal barrier, probably associated with both glycolytic and protein synthesis pathways. Apoptosis may thus play a role in the pathogenesis of intestinal barrier dysfunction.

Animals↗

PubMed1998

Alterations in the functions of the reticuloendothelial and protease-antiprotease systems after intraperitoneal injection of zymosan in rats.

OBJECTIVE: To evaluate alterations in the function of the reticuloendothelial system (RES) and potential protective effects of pretreatment with the antioxidants: N-acetyl-L-cysteine (NAC) or dimethyl sulphoxide (DMSO), after intraperitoneal injection of zymosan (0.50 mg/g body weight) in rats. DESIGN: Experimental study. SETTING: University hospital, Sweden. ANIMALS: 81 male Sprague-Dawley rats. INTERVENTION: Intraperitoneal injection of either 4 ml saline or zymosan suspension (0.50 mg/g body weight). One hour before the intraperitoneal injection, 1 ml of saline, or a solution of NAC (150 mg/kg) or DMSO (80 mg/kg) were given intravenously. MAIN OUTCOME MEASURES: Systemic arterial pressure, packed cell volume, concentrations of plasma proteins and plasma protease inhibitors, uptake of 125I-labelled Escherichia coli in organs, blood clearance and body uptake rate of radiolabelled E. coli, and blood flow in organs at 3, 6, and 12 hours after injection. RESULTS: The uptake of radiolabelled E. coli in the liver, spleen and lungs decreased significantly from 3 hours onwards after zymosan challenge (p <(0.05). Blood clearance and body uptake rate also decreased significantly from 3 hours onwards (p < 0.05), but this did not correlate with the reduction in organ blood flow. Significant falls in plasma concentrations of prekallikrein (p < 0.01) and protease inhibitors (p <0.05) suggested possible contact-phase activation and activation of the kallikrein-kinin and fibrinolytic system. Pretreatment with NAC, and to a less extent DMSO, significantly prevented these alterations in RES function. CONCLUSION: Zymosan induced an impairment in RES function that was not initially associated with a reduction in blood flow. Plasma proteolytic activity seems to be involved in the impaired RES function. Pretreatment with NAC or DMSO effectively improved RES function.

Acetylcysteine↗

PubMed1998

Effect of a platelet-activating factor antagonist on pancreatitis-associated gut barrier dysfunction in rats.

Platelet-activating factor (PAF) may play a critical and primary role in the pathogenesis of acute pancreatitis and pancreatitis-associated distant organ injury. The present study evaluated the effect of a PAF antagonist, lexipafant (an (S)-4-methyl-2[methyl-imidazo[4,5-c]pyridin-1-ylmethyl)-benzene sulphonyl]-amino]pentanoic acid ethyl ester, BB-882; British Biotech Ltd.), on the potential prevention of gut barrier dysfunction, by measuring gut origin sepsis, bidirectional permeability of the intestinal barrier, and pancreatic capillary endothelial barrier integrity, in acute pancreatitis induced by intraductal infusion of 5% sodium taurodeoxycholate. Pancreatic endothelial permeability significantly increased in animals with acute pancreatitis, whereas pretreatment with lexipafant had a preventive effect (p < 0.05 vs. pancreatitis with saline). Similarly, alterations noted in hematocrit and plasma levels of lipase and calcium were counteracted by the PAF antagonist. It also prevented the increase in albumin leakage from blood to the mucosal interstitium and from blood to the intestinal lumen in acute pancreatitis. Albumin passage from the gut lumen to blood in animals with pancreatitis pretreated with saline increased from 3 h and on, and lexipafant prevented alterations in mucosal epithelial permeability. Bacterial translocation was commonly seen in pancreatitis, whereas only a few positive cultures were observed in pancreatitis animals given lexipafant. Microthrombosis in intestinal villi seemed less frequent after lexipafant pretreatment. We conclude that (a) PAF may play a role in the pathogenesis of pancreatitis-associated intestinal dysfunction, (b) PAF may be involved in the development of distant organ dysfunction by triggering endothelial barrier dysfunction, and (c) PAF antagonists may provide potential agents for preventing pancreatitis-associated gut barrier dysfunction.

Acute Disease↗

PubMed1998

The influence of intestinal ischemia and reperfusion on bidirectional intestinal barrier permeability, cellular membrane integrity, proteinase inhibitors, and cell death in rats.

Intestinal ischemia and reperfusion injury (I/R) is probably involved in the pathogenesis of intestinal barrier dysfunction, associated with the concomitant translocation of enteric bacteria and toxins and the potential development of multiple organ failure. The intestinal endothelial and epithelial layers play a major role preventing the entry of toxic substances from the gut, but the influence of protease-antiprotease systemic balance on these barrier functions and the relationship between epithelial DNA synthesis, apoptosis, and endothelial and epithelial barrier macromolecule permeability are not fully investigated. Endothelial and epithelial barrier macromolecular permeability, epithelial DNA synthesis, the endothelial and epithelial plasma membrane system, apoptosis and oncosis, plasma levels of proteinase inhibitors, and proenzymes were measured in rats subjected to 20 and 40 min intestinal ischemia and 1, 3, 6, or 12 h reperfusion. Endothelial permeability increased after both 20 and 40 min intestinal ischemia. Epithelial permeability significantly increased during 1-6 h reperfusion after 20 min ischemia and during 1-12 h reperfusion after 40 min ischemia. Epithelial DNA synthesis increased in animals with 20 min ischemia followed by 12 h reperfusion. Plasma levels of prekallikrein, C1-esterase inhibitor, and alpha1-macroglobulin were significantly lower following both 20 and 40 min ischemia from 3 h reperfusion and on. Apoptotic epithelial cells significantly increased in animals subjected to 20 min ischemia followed by 12 h reperfusion. The severity of reperfusion injury in the intestinal endothelial and epithelial barrier seems to correlate with the period of ischemia and the pathway of cell damage and death, together with proteinase-antiproteinase imbalance.

Albumins↗

PubMed1998

Kartagener's syndrome: a re-visit with Chinese perspectives.

The objective of this study was to evaluate the clinical, radiological investigation profiles, and ciliary function and ultrastructure in Chinese patients with Kartagener's syndrome (presence of dextrocardia, sinusitis and bronchiectasis). All patients with dextrocardia were assessed for the presence of sinusitis and bronchiectasis in our hospital network. Patients identified with Kartagener's were assessed when they were at steady state for their bronchiectasis. Seven cases (4 males; mean age 34.9 years) were identified and systematically reviewed. The mean 24 h sputum volume was 26.6 +/- 32.77 mL/day and the patients suffered from a mean of 2.9 exacerbations/year. Nasal symptoms (anosmia in one, obstruction in six and persistent discharge in three patients) were common. Only two cases (1 M) were married and both had normal fertility. Lung function assessment showed a mean FEV1/FVC of 83.3 +/- 38.78/86.5 +/- 36.72 (% predicted) with little reversibility. High resolution computerized tomography (HRCT) revealed bronchiectactic involvement of the lower lobes in seven and middle lobe/lingula in four cases. Assessment of alpha-1-anti-trypsin, aspergillus precipitins, auto-antibodies and serology for Pseudomonas pseudomallei was normal. Sputum culture yielded Pseudomonas aeruginosa in three, Haemophilus influenzae in three and commensals in one case. Phase contrast microscopy assessment of respiratory cilia, obtained by brushing the inferior turbinate, revealed that most of the mucosa was unciliated. The mean ciliary beat frequency was 5.2 +/- 6.76 Hz (range 0-13.7; normal range 12-18 Hz). Four patients had immotile cilia whilst the rest had normal ciliary movement. Transmission electron microscopy showed the absence of dynein arms in four patients. The results of this study show that patients with Kartagener's syndrome may have normal ciliary ultrastructure and the absence of dynein arms is not necessarily associated with ciliary immotility. The presence of ciliary immotility might have prognostic value as these patients appear to have more active bronchiectasis. Our experience on this series should help clinicians in the investigation and management of these patients.

Adolescent↗

PubMed1998

Dentin-specific proteins in MDPC-23 cell line.

Only four established odontoblast-like cell lines have been reported in the literature (1-6). Of the four, only two synthesize dentin-specific proteins. These studies report that the cell line MO6-G3 synthesizes phosphophoryn (DPP), dentin sialoprotein (DSP) and dentin matrix protein-1 (DMP-1) while MDPC-23 synthesizes DSP, but not DMP-1. The objective of the present study was to determine whether polyclonal antibodies to rat DSP and DPP would label odontoblasts on microscopic sections of day-19 fetal mouse incisor odontoblasts as well as cultured cells of the MDPC-23 cell line. The spontaneously immortalized MDPC-23 cell line was derived from fetal mouse molar papillae, made continuous by the 3T6 method and cloned by dilution. These cultures have been passaged 77 times after cloning, form multilayered nodules, and have high alkaline phosphatase activity. The data show positive reactivity in odontoblasts in 19-d mouse fetal incisors as well as in cultures of MDPC-23 cells by fluorescence and confocal microscopy. In addition, these cultures were characterized by phase microscopy and scanning and transmission electron microscopy. These findings suggest that MDPC-23 cells are of the odontoblast lineage.

Animals↗

PubMed1998

Role of intestinal permeability in monitoring mucosal barrier function. History, methodology, and significance of pathophysiology.

The intestinal barrier function is considered to play an important role in protecting the penetration of luminal antigens, associated with the development of secondary infection and sepsis and the initiation of the multiple organ dysfunction syndrome. The intestinal mucosal barrier against luminal macromolecules and microorganisms consists of both non-immunological and immunological defence mechanisms. The main constituents of the intestinal barrier are the endothelial and epithelial barriers. The epithelial barrier selectively restricts micromolecular permeation and almost completely restricts macromolecular permeation, while the endothelial barrier has a very limited restriction to micromolecules and only partly to macromolecules. Maintenance of the barrier depends on the integrity of cellular plasma membranes and tight junctions, as well as the elaboration of endothelial and epithelial secretory products. Focal denudation of the barrier results in permeation of potentially threatening luminal compounds including antigens, proteases, H+, bacteria and endotoxin, and also other factors chemotactic for inflammatory cells. By initiating inflammation and thus acting on subepithelial tissues, such factors can further influence endothelial and epithelial transport and barrier function. The repair of endothelial and epithelial injury is also complex, and both restitution and enhanced endothelial and epithelial cell proliferation are likely to be important. At present, however, mechanisms for intestinal epithelial and endothelial permeation, their alterations in disease and potential ways to prevent or repair injury, are still not fully elucidated.

Animals↗

PubMed1997

Frequent abnormalities of TSG101 transcripts in human prostate cancer.

TSG101 has been identified as a candidate tumor suppressor gene and abnormal transcripts have been identified in a substantial fraction of breast cancers. To determine whether TSG101 expression is commonly altered in other tumors, a series of 15 primary and metastatic prostate cancers were analysed by reverse transcriptase-PCR amplification. Abnormal transcripts with extensive deletions in the coding region were found in nine of these tumors, while only the normal transcript was found in control and benign prostatic hypertrophy tissues. More than one abnormal transcript was found in four of these nine cases and distinct abnormal TSG101 transcripts were found in separate biopsies taken from one tumor. Importantly, the normal TSG101 transcript was undetectable in two metastatic prostate cancers, indicating the absence of TSG101 protein. Sequence analysis demonstrated that there were at least six distinct deletions, with four of these deletions found in more than one tumor sample. The most commonly identified deletion, from bp 153 to 1055, was identical to a deletion reported previously in breast cancer. These results demonstrate that TSG101 transcripts are frequently abnormal in prostate cancer and suggest that loss of TSG101 protein contributes to disease development or progression.

Base Sequence↗

PubMed1997

Identification of a flexible loop region (297-313) of urokinase-type plasminogen activator, which helps determine its catalytic activity.

Pro-urokinase has a much higher intrinsic catalytic activity than other zymogens of the serine protease family. Lys300(c143) in an apparent "flexible loop" region (297-313) was previously shown to be an important determinant of this intrinsic catalytic activity. This was related to the loop allowing the positive charge of Lys300(c143) to transiently interact with Asp355(c194), thereby inducing an active conformation of the protease domain (Liu, J. N., Tang, W., Sun, Z., Kung, W., Pannell, R., Sarmientos, P., and Gurewich, V. (1996) Biochemistry 35, 14070-14076). To further test this hypothesis, the charge at position 300(c143) and the flexibility of the loop were altered using site-directed mutagenesis designed according to a computer model to affect the interaction between Lys300(c143) and Asp355(c194). When the charge at Lys300(c143) but not Lys313(c156) was reduced, a significant reduction in the intrinsic catalytic activity occurred. Similarly, when the flexibility (wobbliness) of the loop was enhanced reducing the size of side chain, the intrinsic catalytic activity was also reduced. By contrast, when the loop was made less flexible, the intrinsic catalytic activity was increased. These findings were consistent with the hypothesis. The effects of these mutations on two-chain activity were less and often discordant with the intrinsic catalytic activity, indicating that they can be modulated independently. This structure-function disparity can be exploited to create a more zymogenic pro-urokinase (lower intrinsic catalytic activity) with a high catalytic activity, as exemplified by two of the mutants. The changes in intrinsic catalytic activity and two-chain activity induced by the mutations were due to changes in kcat rather than Km. Some significant structure-function differences between pro-urokinase and its highly homologous counterpart, tissue plasminogen activator, were also found.

Catalysis↗