Biomedical subjects

D H Shin

Publications and source records attributed to D H Shin.

At least 127 records · Page 7Linked to original sources

PubMed1998

Effect of topical anti-inflammatory treatment on the long-term outcome of laser trabeculoplasty. Fluorometholone-Laser Trabeculoplasty Study Group.

PURPOSE: To investigate the effect of anti-inflammatory treatment on the long-term outcome of argon laser trabeculoplasty, we extended the short-term study of the Fluorometholone-Laser Trabeculoplasty Study Group. METHODS: Among the chronic open-angle glaucoma patients randomly assigned to the 0.25% fluorometholone or placebo eyedrops four times daily before and after argon laser trabeculoplasty in the Fluorometholone-Laser Trabeculoplasty Study, the long-term records of 27 patients (13 eyes in the fluorometholone group and 14 eyes in the placebo group) were available for our review and analysis. RESULTS: There were no statistically significant differences in the success rate of argon laser trabeculoplasty between the fluorometholone and control groups over the follow-up period of 4.6 +/- 3.4 years. CONCLUSION: While it decreased ocular inflammation and discomfort during the early postoperative period, peri-argon laser trabeculoplasty use of a topical corticosteroid did not influence the long-term intraocular pressure outcome of argon laser trabeculoplasty.

Administration, Topical↗

PubMed1998

Outcomes of primary phakic trabeculectomies without versus with 0.5- to 1-minute versus 3- to 5-minute mitomycin C.

PURPOSE: To compare the intraocular pressure and hypotony outcomes of primary phakic trabeculectomies with no mitomycin C (MMC), shorter MMC, and longer MMC exposure. METHODS: We evaluated primary phakic trabeculectomies with no MMC (36 eyes of 36 patients), 0.5- to 1-minute MMC (50 eyes of 50 patients), and 3- to 5-minute MMC (38 eyes of 38 patients) at the concentration of 0.5 mg/ml. Successful trabeculectomy was defined as an intraocular pressure of 21 mm Hg or less without development of a marked visual acuity loss associated with prolonged hypotony (intraocular pressure < 6 mm Hg over 3 months) and without the need for additional surgery to control intraocular pressure or treat postoperative complications. RESULTS: The three groups were similar in demographics, preoperative intraocular pressure, and medical dependency. However, the incidence of hypotony during the postoperative periods of 3 to 12 months was significantly higher in the 3- to 5-minute MMC group (P < .05, chi-square test). Severe visual acuity loss associated with hypotony was also more frequently found in the 3- to 5-minute MMC group than in the 0.5- to 1-minute (P = .009, chi-square test) group or the control group (P = .014, chi-square test). In addition, the success probabilities were significantly different among the three groups (P = .001, Kaplan-Meier survival analysis with log-rank test) and were the highest in the 0.5- to 1-minute MMC group and the lowest in the 3- to 5-minute MMC group. CONCLUSION: Shorter application (0.5 to 1 minute) of MMC appears to be optimal for the successful outcome of primary phakic trabeculectomy compared with no MMC or longer application of MMC at a concentration of 0.5 mg/ml.

Aged↗

PubMed1998

Limbus-based vs fornix-based conjunctival flap in combined glaucoma and cataract surgery with adjunctive mitomycin C.

PURPOSE: To determine the efficacy and safety of limbus-based vs fornix-based conjunctival flaps in patients with primary open-angle glaucoma undergoing trabeculectomy combined with phacoemulsification and intraocular lens implantation with adjunctive subconjunctival mitomycin C. METHODS: In a prospective study, 69 eyes of 69 patients with primary open-angle glaucoma, visually symptomatic cataracts, and no previous incisional ocular surgery were randomly assigned to limbus-based and fornix-based conjunctival flap groups. All patients received trabeculectomy combined with phacoemulsification and posterior chamber lens implantation with 1-minute (0.5 mg/ ml) application of subconjunctival mitomycin C. RESULTS: The mean intraocular pressures were significantly (P < .05) lower on significantly (P < .05) fewer medications postoperatively at 1 week, 1 month, 3, 6, 9, 12, and 15 to 18 months, and at last follow-up in both groups than they had been preoperatively. However, there were no significant (P > .05) differences in postoperative mean intraocular pressure, mean number of medications, and visual acuity between the two groups at any time interval. Hypotony with wound leak was significantly (P = .019) higher in the limbus-based group. Other postoperative complications were not significantly (P > .05) different between the two groups. CONCLUSIONS: There was no notable difference in glaucoma control or visual outcome between limbus-based and fornix-based conjunctival flaps in primary trabeculectomy combined with phacoemulsification and lens implantation with adjunctive subconjunctival mitomycin C. The fornix-based flap was as safe as, if not safer than, the limbus-based flap in the glaucoma triple procedure with adjunctive subconjunctival mitomycin C.

Aged↗

PubMed1998

Primary glaucoma triple procedure in patients with primary open-angle glaucoma: the effect of mitomycin C in patients with and without prognostic factors for filtration failure.

PURPOSE: To investigate the effect of adjunctive mitomycin C on primary glaucoma triple procedure in patients with primary open-angle glaucoma with and without one or more of the prognostic factors for filtration failure of primary glaucoma triple procedure. Those factors include being of African-American race, having a preoperative intraocular pressure of 20 mm Hg or more on maximum tolerated medications, and being on two or more medications preoperatively. METHODS: Study patients consisted of 197 consecutive patients with primary open-angle glaucoma who were randomly assigned to receive either no adjunctive mitomycin C (101 eyes of 101 patients) or to receive adjunctive subconjunctival mitomycin C (96 eyes of 96 patients) during the primary glaucoma triple procedure. Kaplan-Meier survival analysis comparisons were made between respective subgroups with and without prognostic indicators for filtration failures using a relatively stringent set of criteria for filtration success of primary glaucoma triple procedure. RESULTS: There was no statistically significant (P = .117) difference in filtration success of primary glaucoma triple procedure between the control and mitomycin C groups. Adjunctive mitomycin C significantly (P < .05) improved the filtration outcome of the primary glaucoma triple procedure in the subgroups with each of the three prognostic factors for filtration failure of primary glaucoma triple procedure. On the other hand, in the subgroups without the prognostic factors, adjunctive mitomycin C did not significantly (P > .05) change the filtration outcome of the primary glaucoma triple procedure. CONCLUSION: These findings establish the basis for selective use of mitomycin C in patients with primary open-angle glaucoma undergoing primary glaucoma triple procedure.

Aged↗

PubMed1998

Q-switched 532-nm Nd:YAG laser trabeculoplasty (selective laser trabeculoplasty): a multicenter, pilot, clinical study.

OBJECTIVE: To investigate the safety and efficacy of a new laser procedure using a q-switched 532-nm neodymium (Nd):YAG laser, also called "selective laser trabeculoplasty," to lower intraocular pressure (IOP) in patients with open-angle glaucoma (OAG). The laser parameters were set to selectively target pigmented trabecular meshwork (TM) cells without coagulative damage to the TM structure or nonpigmented cells. DESIGN: Nonrandomized, prospective, clinical trial. PARTICIPANTS: Thirty eyes of 30 patients with uncontrolled OAG (OAG group) and 23 eyes of 23 patients with uncontrolled OAG treated previously with argon laser trabeculoplasty (ALT group) were observed for 4 to 26 weeks. Forty-four of the 53 eyes were observed for 26 weeks. INTERVENTION: Patients were treated with the Coherent Selecta 7000 (Coherent, Inc, Palo Alto, CA) frequency-doubled q-switched Nd:YAG laser (532 nm). A total of approximately 50 nonoverlapping spots were placed over 180 degrees of the TM at energy levels ranging from 0.6 to 1.2 mJ per pulse. After surgery, patients were maintained with the identical drug regimen as that before treatment. RESULTS: Both the OAG and ALT groups showed similar IOP reductions over time. Seventy percent of patients in each group responded to treatment with an IOP reduction of least 3 mmHg. At 26 weeks of follow-up, mean IOP reduction was 5.8 mmHg (23.5%, P < 0.001) for the OAG group and 6.0 mmHg (24.2%, P < 0.001) for the ALT group. The untreated eye showed a 9.7% (P < 0.001) reduction of IOP at 26 weeks. However, the IOP difference between the treated and untreated eyes was statistically significant at P < 0.003. Transient IOP elevation of 5 mmHg or greater was seen in 24% of patients. CONCLUSION: The selective laser trabeculoplasty appears to be a safe and effective method to lower IOP in patients with OAG and patients treated previously with ALT. A reduction of IOP can be achieved without coagulation of the TM.

Adult↗

PubMed1998

Topical verapamil and episcleral venous pressure.

OBJECTIVE: To investigate the effect of a single-dose and 2-week administration of topical 0.25% verapamil on episcleral venous pressure (EVP) and intraocular pressure (IOP). DESIGN: A prospective, randomized, double-masked, crossover, and placebo-controlled study. PARTICIPANTS: One eye of each of 20 normal subjects received topical verapamil or placebo. INTERVENTION: The EVP was measured using a pressure chamber episcleral venomanometer. The IOP, EVP, heart rate (HR), and blood pressure (BP) were measured at baseline, 1.5 hours after a single drop, and after 2-week, three-times-a-day topical administration of 0.25% verapamil or placebo to one eye. After a 4-week washout period, the treatment to the study eye was reversed, and measurements of EVP, IOP, HR, and BP were repeated. MAIN OUTCOME MEASURES: The EVP, IOP, HR, and BP were measured. RESULTS: Ninety minutes after a single drop, the EVP in the verapamil-treated eyes was reduced by 4.8% (P = <0.001) and in the untreated fellow eyes by 2.4% (P = 0.032). After 2-week topical administration, the EVP was reduced by 9.5% (P = <0.0001) in the verapamil-treated eyes and by 4.8% (P = 0.001) in the fellow eyes. The EVP was unchanged in the treated and fellow eyes after a single drop or after a 2-week treatment with placebo. Ninety minutes after topical administration of 0.25% verapamil, the IOP decreased by 7.7% (P = <0.0001) in the treated eyes and decreased by 3.6% (P = 0.03) in the fellow eyes. After a 2-week administration of topical verapamil, the IOP decreased by 12% (P = 0.0001) in the treated eyes and by 7.2% (P = 0.0289) in the fellow eyes. The changes of IOP in the placebo-treated and fellow eyes were not statistically significant after a single-dose and 2-week administration. After topical administration of verapamil, a contralateral effect on IOP and EVP was observed in the fellow, untreated eye. No systemic effect on HR or BP was detected after a single-dose administration of topical verapamil. There were, however, significant reductions in HR and BP after a 2-week treatment with topical 0.25% verapamil. CONCLUSION: These results indicate that a single drop and a 2-week administration of topical 0.25% verapamil decrease IOP and EVP significantly, with more pronounced reduction after a 2-week treatment than after a single-dose treatment.

Administration, Topical↗

PubMed1998

The role of adjunctive mitomycin C in secondary glaucoma triple procedure as compared to primary glaucoma triple procedure.

OBJECTIVE: This study aimed to investigate whether previously failed glaucoma filtration surgery is a risk factor for filtration failure of subsequent trabeculectomy combined with cataract surgery and to determine the role of adjunctive mitomycin C (MMC) in the secondary glaucoma triple procedure (SGTP) as compared to primary glaucoma triple procedure (PGTP). DESIGN: A prospective, controlled study that was randomized with respect to assignment to adjunctive MMC and a case-control design with respect to comparisons between SGTP and PGTP was studied. PARTICIPANTS: The SGTP group consisted of 49 eyes of 49 consecutive patients with primary open-angle glaucoma with a history of glaucoma filtration surgery requiring glaucoma medical therapy and in need of cataract surgery, randomized to adjunctive MMC (SGTP MMC subgroup of 21 eyes) and no adjunctive MMC (SGTP control subgroup of 28 eyes). The PGTP group consisted of 49 PGTP cases closely matched to the SGTP cases with respect to age, race, gender, MMC use, C:D ratio, and systemic diseases. INTERVENTION: Trabeculectomy combined with phacoemulsification and a small incision (5 x 6 mm), all polymethylmethacrylate posterior chamber intraocular lens implantation with or without adjunctive MMC (0.5 mg/ml for 1 minute), was performed. MAIN OUTCOME MEASURES: Surgery failure was defined as the need of an additional intraocular procedure or the need of more than one medication to achieve intraocular pressure control to the target level. Intragroup and intergroup comparisons were made with respect to filtration outcome among the SGTP and PGTP patients. RESULTS: Without adjunctive MMC, filtration success was significantly less in SGTP than in PGTP (P = 0.03). Adjunctive MMC significantly increased the success rate of SGTP (P = 0.02) but not that of PGTP (P = 0.89) over the average follow-up period of 2 years. CONCLUSIONS: Previously failed glaucoma filtration surgery is a significant risk factor for the filtration failure of combined surgery. Intraoperative use of adjunctive MMC significantly improves the filtration success rate of SGTP.

Adult↗

PubMed1998

Decrease of capsular opacification with adjunctive mitomycin C in combined glaucoma and cataract surgery.

OBJECTIVE: The authors investigated the incidence of capsular opacification requiring YAG capsulotomy after primary trabeculectomy combined with phacoemulsification and implantation of all polymethylmethacrylate intraocular lenses. DESIGN: A prospective randomized study. PARTICIPANTS: One hundred seventy-four eyes of 174 nonselected patients with primary open-angle glaucoma (POAG) were randomized to either no adjunctive mitomycin C (MMC) control group of 93 eyes of 93 patients) or adjunctive subconjunctival MMC (MMC group of 81 eyes of 81 patients) during the primary glaucoma triple procedure (PGTP). INTERVENTION: Primary glaucoma triple procedure with and without MMC and YAG laser capsulotomy for posterior capsular opacification (PCO) was performed. MAIN OUTCOME MEASURES: The incidences of YAG capsulotomy for PCO were compared between the control and MMC groups and also between the control group and the MMC subgroups (1 minute, 3 minutes, and 5 minutes of MMC application) using Kaplan-Meier analysis with Mantel-Cox log-rank test. Cox proportional hazard regression analysis also was performed to identify significant factors affecting capsular opacification. RESULTS: The control and MMC groups were similar in preoperative characteristics. However, the probability of PCO requiring YAG capsulotomy was significantly lower in the MMC group than in the control group (P = 0.004). Among the MMC subgroups, MMC application for 3 minutes was most effective and significant when compared with that of the control group (P = 0.002). Although not as significant as the intraoperative use of MMC (P = 0.002), old age (P = 0.026) and presence of diabetes mellitus (P = 0.035) were also identified as significant beneficial factors for decreasing the incidence of YAG capsulotomy for PCO in Cox proportional hazard regression analysis. CONCLUSION: Intraoperative subconjunctival MMC application during combined glaucoma and cataract surgery has a beneficial effect of inhibiting PCO after combined surgery in patients with POAG. Thus, after intraoperative subconjunctival application of MMC at the concentration of 0.5 mg/ml for 3 minutes, the aqueous MMC level must have been great enough to inhibit the lens epithelial cell proliferation to result in a long-term decrease in PCO.

Aged↗

PubMed1998

Corneal topography in Ehlers-Danlos syndrome.

PURPOSE: To assess the use of corneal topography in conjunction with slitlamp biomicroscopy and retinoscopy to diagnose keratoconus in a large group of patients with Ehlers-Danlos syndrome (EDS). SETTING: Kresge Eye Institute, Wayne State University, Detroit, Michigan, USA. METHODS: Thirty-six patients (72 eyes) with genetically typed EDS had slitlamp biomicroscopy, retinoscopy, and videokeratography with the EyeSys instrument. The presence or absence of slitlamp keratoconus findings was correlated to a presumptive diagnosis based on corneal topography using derived topographic indexes associated with keratoconus. These topographic indexes included central corneal power, (CCP), difference in CCP, inferosuperior asymmetry (I-S) value, and asphericity (Q). Axial and profile difference maps were generated and analyzed for findings suggestive of keratoconus. RESULTS: In 72 eyes, no keratoconus was found using slitlamp biomicroscopy. No eye had an I-S value greater than 1.60 diopters (D), 2 eyes had a CCP greater than 46.50 D, and 2 eyes had a Q value less than -1.00. Eight of 36 pairs of eyes had an intereye CCP greater than 0.92 D. In both eyes of the patient with Q values less than -1.00 the profile difference maps were mildly abnormal. CONCLUSIONS: Slitlamp biomicroscopy of the cornea was unremarkable in all patients. Only 1 patient had Q values and profile difference maps that were mildly suggestive of keratoconus. Even after adding topography to the examination, it appears that keratoconus in a known population of patients with EDS remains rare.

Cornea↗

PubMed1998

Cloning and bacterial expression of sesquiterpene cyclase, a key branch point enzyme for the synthesis of sesquiterpenoid phytoalexin capsidiol in UV-challenged leaves of Capsicum annuum.

Sesquiterpene cyclase, a branch point enzyme in the general isoprenoid pathway for the synthesis of phytoalexin capsidiol, was induced in detached leaves of Capsicum annuum (pepper) by UV treatment. The inducibility of cyclase enzyme activities paralleled the absolute amount of cyclase protein(s) of pepper immunodetected by monoclonal antibodies raised against tobacco sesquiterpene cyclase. A cDNA library was constructed with poly(A)+ RNA isolated from 24 h UV-challenged leaves of pepper. A cDNA clone for sesquiterpene cyclase in pepper was isolated by using a tobacco 5-epi aristolochene synthase gene as a heterologous probe. The predicted protein encoded by this cDNA was comprised of 559 amino acids and had a relative molecular mass of 65,095. The primary structural information from the cDNA clone revealed that it shared 77%, 72% and 49% identity with 5-epi aristolochene, vetispiradiene, and cadinene synthase, respectively. The enzymatic product catalyzed by the cDNA clone in bacteria was identified as 5-epi aristolochene, as judged by argentation TLC. RNA blot hybridization demonstrated the induction of an mRNA consistent with the induction of cyclase enzyme activity in UV-treated pepper.

Amino Acid Sequence↗

PubMed1998

Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis.

Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.

Amino Acid Sequence↗

PubMed1998

[A study on the movement for establishing a private isolation hospital under the rule of Japanese imperialism].

Koreans had an ill feeling against the only public isolation hospital, and their ill feeling was a cause the movement for establishing a private isolation hospital. The bad seating capacity and the nearness to downtown of public isolation hospital, and some violence caused in the course of taking a patient to the public isolation hospital were concrete causes of the movement for establishing a private isolation hospital. The three purposes of the movement for establishing a private isolation hospital are as follows. First, for the purpose of removing the national ill feeling, Koreans had to establish the isolation hospital by themselves. Second, for the purpose of curing the patients, doctors needed to use Oriental medicine together. Third, for the purpose of doing away with the anxiety of infection, the isolation hospital had to be located in a distant place from downtown. The movement for establishing a private isolation hospital didn't succeed. It only ended up establishing a contagious ward in Severance hospital. Because whenever an isolation hospital was planned to be constructed somehere, the residents worrying about the infection opposed to the construction, and enough money didn't be gathered. Abve all, rich pro-Japanese men didn't contribute enough money. The middle and lower classes contributed almost all of the money. The movement for estabishing a private isolation hospital was the extension of anti-Japanese national feeling that sprung from the March 1st movement. In view of strenghtening Korean ability, the movement for establishing a private isolation hospital had a common cause with the Shilryokyangsong movement in the early 1920s.

Cholera↗

PubMed1998

Comparison between high-pass resolution perimetry and differential light sensitivity perimetry in patients with glaucoma.

PURPOSE: High-pass resolution perimeters such as the Ophthimus (High Tech Vision, Malmö, Sweden) may provide an easier, faster assessment of the visual field than conventional perimeters such as the Humphrey (Humphrey Instruments Inc., San Leandro, CA, U.S.A.), which is based on differential light sensitivity. The Ophthimus provides global indices and statistical analyses conceptually similar to those produced by the Humphrey. In addition, the Ophthimus provides, as a unique parameter, the estimated neural capacity. PATIENTS AND METHODS: Twenty-one patients with chronic open-angle glaucoma and reliable fields on both Humphrey and Ophthimus testing were evaluated. The results from the two different techniques were compared. RESULTS: The estimated neural capacity was correlated to global deviation and mean deviation at a statistically significant level. There were also correlations between mean deviation and global deviation and between pattern standard deviation and local deviation, but not between retest standard deviation and short-term fluctuation. CONCLUSION: It is not clear at this point if the neural capacity of the Ophthimus provides any clinically useful information over and above global deviation. Mean test time was shorter with the Ophthimus system than the Humphrey. Performance on the reliability indices was better for the Ophthimus. The Ophthimus may prove useful in following patients with glaucoma.

Aged↗

PubMed1998

Use of autologous Tenon's capsule and scleral patch grafts for repair of excessively draining fistulas with leaking filtering blebs.

PURPOSE: To report the use of autologous Tenon's and partial-thickness scleral tissues for patch graft revisions of excessively draining fistulas associated with leaking filtering blebs. METHODS: Appropriate sized pieces of autologous Tenon's tissue and partial-thickness scleral tissue were dissected near the surgical sites and used as patch grafts to effectively repair excessively draining fistulas with leaking filtering blebs. RESULTS: Both Tenon's and partial-thickness scleral patch grafts were useful in adequately closing the excessively draining fistulas. CONCLUSION: Autologous Tenon's and scleral tissue patch grafts appear to be safe and effective for the repair of excessively draining fistulas, and are useful when patch grafts are unexpectedly needed.

Aged↗

PubMed1997

Crystal structure of carboxylesterase from Pseudomonas fluorescens, an alpha/beta hydrolase with broad substrate specificity.

BACKGROUND: A group of esterases, classified as carboxylesterases, hydrolyze carboxylic ester bonds with relatively broad substrate specificity and are useful for stereospecific synthesis and hydrolysis of esters. One such carboxylesterase from Pseudomonas fluorescens is a homodimeric enzyme, consisting of 218-residue subunits. It shows a limited sequence similarity to some members of the alpha/beta hydrolase superfamily. Although crystal structures of a number of serine esterases and lipases have been reported, structural information on carboxylesterases is very limited. This study was undertaken in order to provide such information and to understand a structural basis for the substrate specificity of this carboxylesterase. RESULTS: In this study, the crystal structure of carboxylesterase from P. fluorescens has been determined by the isomorphous replacement method and refined to 1.8 A resolution. Each subunit consists of a central seven-stranded beta sheet flanked by six alpha helices. The structure reveals the catalytic triad as Ser 114-His 199-Asp 168. The structure of the enzyme in complex with the inhibitor phenylmethylsulfonyl fluoride has also been determined and refined to 2.5 . The inhibitor is covalently attached to Ser 114 of both subunits, with the aromatic ring occupying a hydrophobic site defined by the aliphatic sidechains of Leu23, Ile58, Ile70, Met73 and Val170. No large structural changes are observed between the free and inhibitor-bound structures. CONCLUSIONS: Carboxylesterase from P. fluorescens has the alpha/beta hydrolase fold and the Ser-His-Asp catalytic triad. The active-site cleft in each subunit is formed by the six loops covering the catalytic serine residue. Three of the active-site loops in each subunit are involved in a head-to-head subunit interaction to form a dimer; it may be these extra structural elements, not seen in other esterases, that account for the inability of carboxylesterase to hydrolyze long chain fatty acids. As a result of dimerization, the active-site clefts from the two subunits merge to form holes in the dimer. The active-site clefts are relatively open and thus the catalytic residues are exposed to the solvent. An oxyanion hole, formed by nitrogen atoms of Leu23 and Gln115, is present in both the free and inhibitor-bound structures. An open active site, as well as a large binding pocket for the acid part of substrates, in P. fluorescens carboxylesterase may contribute to its relatively broad substrate specificity.

Anions↗

PubMed1997

The heat-shock protein HslVU from Escherichia coli is a protein-activated ATPase as well as an ATP-dependent proteinase.

HslVU in Escherichia coli a new two-component ATP-dependent protease composed of two heat-shock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome beta-type subunits. Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B-chain, casein, and carboxymethylated lactalbumin. Maximal proteolytic activity was obtained with a 1:2 molar ratio of HslV (a 250-kDa complex) to HslU (a 450-kDa complex). By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20-fold by HslU in the presence of ATP. The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2-3-fold in the presence of HslV. Concentrations of insulin B-chain that maximally stimulated the ATPase allowed maximal rates of the B-chain hydrolysis. Furthermore, addition of increasing amounts of ADP or N-ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel. Thus, HslVU is a protein-activated ATPase as well as an ATP-dependent proteinase, and these processes appear linked. Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis. Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins. Thus, the functional requirements for ATP-dependent hydrolysis of peptides and proteins appear different.

ATP-Dependent Proteases↗

PubMed1997

The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor.

BACKGROUND: . Lipases, a family of enzymes which catalyze the hydrolysis of triglycerides, are widely distributed in many organisms. True lipases are distinguished from esterases by the characteristic interfacial activation they exhibit at an oil-water interface. Lipases are one of the most frequently used biocatalysts for organic reactions performed under mild conditions. Their biotechnological applications include food and oil processing and the preparation of chiral intermediates for the synthesis of enantiomerically pure pharmaceuticals. Recent structural studies on several lipases have provided some clues towards understanding the mechanisms of hydrolytic activity, interfacial activation, and stereoselectivity. This study was undertaken in order to provide structural information on bacterial lipases, which is relatively limited in comparison to that on the enzymes from other sources. RESULTS: . We have determined the crystal structure of a triacylglycerol lipase from Pseudomonas cepacia (PcL) in the absence of a bound inhibitor using X-ray crystallography. The structure shows the lipase to contain an alpha/beta-hydrolase fold and a catalytic triad comprising of residues Ser87, His286 and Asp264. The enzyme shares several structural features with homologous lipases from Pseudomonas glumae (PgL) and Chromobacterium viscosum (CvL), including a calcium-binding site. The present structure of PcL reveals a highly open conformation with a solvent-accessible active site. This is in contrast to the structures of PgL and PcL in which the active site is buried under a closed or partially opened 'lid', respectively. CONCLUSIONS: . PcL exhibits some structural features found in other lipases. The presence of the Ser-His-Asp catalytic triad, an oxyanion hole, and the opening of a helical lid suggest that this enzyme shares the same mechanisms of catalysis and interfacial activation as other lipases. The highly open conformation observed in this study is likely to reflect the activated form of the lipase at an oil-water interface. The structure suggests that the interfacial activation of bacterial lipases involves the reorganization of secondary structures and a large movement of the lid to expose the active site. This is similar to the mechanism described for other well characterized fungal and mammalian lipases.

Amino Acid Sequence↗

PubMed1997

GABA metabolism during status epilepticus in the developing rat brain.

The rate of synthesis of GABA, the major inhibitory neurotransmitter, was determined in parietal cortex and hippocampus during SE induced by systemic administration of lithium (3 mEq/kg) followed 20 h later by pilocarpine (100 mg/kg) in 1-4-week-old rats. Our results show that the immature hippocampus is better capable of maintaining GABA synthesis in the face of SE at the earliest stages of development studied (74.1% of basal in 1-week-old) and that development results in a progressive decline in the ability to maintain GABA synthesis in the face of SE (44.1% of basal by 4 weeks) that may parallel the ontogeny of self-sustaining seizures. Our data describe an aspect of developmental GABA neurochemistry which may in part explain the relative resistance of the immature hippocampus to seizure spread and of certain types of seizure-induced damage.

Age Factors↗