Direct demonstration that homotetrameric chaperone SecB undergoes a dynamic dimer-tetramer equilibrium.

We have shown here that the cytosolic bacterial chaperone SecB is a structural dimer of dimers that undergoes a dynamic equilibrium between dimer and tetramer in the native state. We demonstrated this equilibrium by mixing two tetrameric species of SecB that can be distinguished by size. We showed that the homotetrameric species exchanged dimers, because when the mixture was analyzed both by size exclusion chromatography and native polyacrylamide gel electrophoresis a third hybrid tetrameric species was detected. Furthermore, treatment of SecB with 5,5'-dithiobis-(2-nitrobenzoic acid), which modifies the sulfhydryl group on cysteines, caused irreversible dissociation to a dimer indicating that cysteine must be involved in the stabilizing interactions at the dimer interface. It is clear that the two dimer-dimer interfaces of the SecB tetramer are differentially stable. Dissociation at one interface allows for a dynamic dimer-tetramer equilibrium. Because only dimers were exchanged it is clear that the other interface between dimers is significantly more stable, otherwise oligomers should have formed with a random distribution of monomers.

Ketorolac versus prednisolone versus combination therapy in the treatment of acute pseudophakic cystoid macular edema.

OBJECTIVE: To evaluate the efficacy of ketorolac tromethamine 0.5% ophthalmic solution, prednisolone acetate 1.0% ophthalmic solution, and ketorolac and prednisolone combination therapy in the treatment of acute, visually significant, cystoid macular edema (CME) occurring after cataract extraction surgery. DESIGN: Randomized, double-masked, prospective trial. PARTICIPANTS: Twenty-eight patients who had undergone cataract extraction and in whom clinical CME developed within 21 to 90 days after cataract surgery. METHODS: Patients were randomized to topical therapy with ketorolac (group K), prednisolone (group P), or ketorolac and prednisolone combination therapy (group C) four times daily. Treatment was continued until CME resolved or for 3 months, whichever occurred first. Treatment was then tapered over 3 weeks. Examinations were monthly and included Snellen visual acuity, contrast sensitivity, Amsler grid, slit-lamp examination, dilated fundus examination, and fluorescein angiography. RESULTS: Twenty-six of 28 patients completed the study. Patients were enrolled an average of 48 days after surgery. The average improvements in Snellen visual acuity were as follows: 1.6 lines in group K, 1.1 lines in group P, and 3.8 lines in group C. This reached statistical significance for all visits when group C was compared with group P, and for visits 4 and 5 when group C was compared with group K. Group C reached a mean change of two lines or more by visit 2; at no time did either group K or P reach a mean two-line improvement. At no time was a significant difference detected between group K and P with regard to visual acuity or change from baseline. A two-line or more improvement in Snellen acuity was achieved in 16 of 26 patients (61%). Analysis by group revealed four of eight patients (50%) in group P, six of nine patients (67%) in group K, and eight of nine patients (89%) in group C who had achieved a two-line or more improvement. In patients who did improve two lines or more, improvement occurred an average of 2.75 months after initiating therapy in group P, 1.43 months in group K, and 1.33 months in group C. Improvements in contrast sensitivity and leakage on fluorescein angiography tended to mirror improvements in Snellen acuity. CONCLUSIONS: Treatment of acute, visually significant pseudophakic CME with ketorolac and prednisolone combination therapy appears to offer benefits over monotherapy with either agent alone. Patients were more likely to experience recovery of two lines or more of visual acuity. Patients treated with combination therapy or ketorolac monotherapy responded more quickly than did patients treated with prednisolone alone.

Complexes between protein export chaperone SecB and SecA. Evidence for separate sites on SecA providing binding energy and regulatory interactions.

During localization to the periplasmic space or to the outer membrane of Escherichia coli some proteins are dependent on binding to the cytosolic chaperone SecB, which in turn is targeted to the membrane by specific interaction with SecA, a peripheral component of the translocase. Five variant forms of SecB, previously demonstrated to be defective in mediating export in vivo (Gannon, P. M., and Kumamoto, C. A. (1993) J. Biol. Chem. 268, 1590-1595; Kimsey, H. K., Dagarag, M. D., and Kumamoto, C. A. (1995) J. Biol. Chem. 270, 22831-22835) were investigated with respect to their ability to bind SecA both in solution and at the membrane translocase. We present evidence that at least two regions of SecA are involved in the formation of active complexes with SecB. The variant forms of SecB were all capable of interacting with SecA in solution to form complexes with stability similar to that of complexes between SecA and wild-type SecB. However, the variant forms were defective in interaction with a separate region of SecA, which was shown to trigger a change that was correlated to activation of the complex. The region of SecA involved in activation of the complexes was defined as the extreme carboxyl-terminal 21 aminoacyl residues.

Mutational alterations in the homotetrameric chaperone SecB that implicate the structure as dimer of dimers.

Variant forms of SecB with substitutions of aminoacyl residues in the region from 74 to 80 were analyzed with respect to their ability to bind a physiological ligand, precursor galactose-binding protein, and to their oligomeric states. SecBL75Q and SecBE77K are tetramers with affinity for ligand indistinguishable from that of the wild-type SecB, and thus the export defect exhibited by strains producing these variants must result from an effect on interactions between SecB and other components. SecBF74I is tetrameric but binds ligand with a lower affinity. Substitutions at positions 76, 78, and 80 cause a shift in the equilibrium so that the SecB tetramer dissociates into dimers. We conclude that the tetramer is a dimer of dimers and that the residues Cys76, Val78, and Gln80 must be involved either directly or indirectly in forming the interface between dimers. These variant species are defective in binding ligand; however, because their oligomeric state is altered no conclusion can be drawn concerning the direct role of these residues in ligand binding.

Visual and surgical outcomes of retinal detachment following macular hole repair.

OBJECTIVE: To characterize 20 cases of retinal detachment (RD) following surgical repair of macular holes. METHODS: Retrospective review of 20 eyes in 16 patients (4 patients [25%] had bilateral macular hole repairs with subsequent RD) who developed RD in the same eye in which surgical repair of a macular hole had been performed. RESULTS: Twenty detachments in 16 patients were reviewed. The average duration between macular hole repair and presentation of RD was 5.5 weeks. The inferior retina was involved more frequently than the superior retina. A total of 76% of all breaks were located inferiorly. Ten of the 20 eyes were asymptomatic at the time the detachment was diagnosed. Of the 20 eyes, 19 underwent surgical repair, all with anatomic reattachment. At final follow-up, the macular hole was closed in all 20 eyes, and 60% of the patients had final visual acuity improved by 2 lines or more over that before their macular hole repair. CONCLUSION: Retinal detachment is a complication of macular hole surgery. These detachments tend to occur within the first 2 months of follow-up, and have a high success rate of anatomic reattachment with surgery. The occurrence of RD does not preclude improved final visual acuity.

The interaction between the chaperone SecB and its ligands: evidence for multiple subsites for binding.

The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli. It functions to bind and deliver precursors of exported proteins to the membrane-associated translocation apparatus before the precursors fold into their native stable structures. The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands. A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochem Sci 20:65-69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions. Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist.

Silicone oil in the repair of complex retinal detachments. A prospective observational multicenter study.

OBJECTIVE: This study aimed to report anatomic and visual acuity outcomes and complications after 1000-centistoke silicone oil was used as a retinal tamponade for the treatment of complex retinal detachments. DESIGN: Prospective observational multicenter study conducted at community and university-based ophthalmology clinics. PARTICIPANTS: The study cohort consisted of 2439 patients (2573 eyes) treated for complex retinal detachments associated with cytomegalovirus (CMV) necrotizing retinitis or a non-CMV etiology, including proliferative diabetic retinopathy, giant retinal tears, proliferative vitreoretinopathy, or ocular trauma. INTERVENTION: Vitrectomy surgery was performed for complex retinal detachment with 1000-centistoke silicone oil as the retinal tamponade. MAIN OUTCOME MEASURES: Anatomic outcomes were complete retinal attachment and macular attachment. Visual acuity outcomes were ambulatory vision (> or = 4/200) and preservation of preoperative visual acuity. Complications were rates of secondary intraocular pressure elevation (> or = 30 mmHg), hypotony (< or = 5 mmHg), corneal opacification (including band keratopathy, corneal edema, and corneal abrasions), oil emulsification, and cataract. Outcomes were assessed 6, 12, and 24 months after surgery. RESULTS: At the 6-month examination, the retina was completely attached in 178 (78%) of 228 CMV eyes and in 855 (70%) of 1219 non-CMV eyes. The macula was attached in 216 (95%) of 228 and 1062 (89%) of 1189 CMV and non-CMV eyes, respectively. Ambulatory vision was noted in 151 (65%) of 234 CMV eyes and in 480 (38%) of 1251 non-CMV eyes. Visual acuity was preserved in 106 (46%) of 230 and 1035 (84%) of 1229 CMV and non-CMV eyes, respectively. The corresponding rates of complications for CMV and non-CMV eyes were: elevated intraocular pressure, 0 (0%) of 196 and 35 (3%) of 1196; hypotony, 11 (6%) of 196 and 228 (19%) of 1196; corneal opacity, 13 (6%) of 229 and 326 (26%) of 1248; emulsification, 3 (1%) of 211 and 29 (3%) of 959; and cataract in phakic eyes, 118 (64%) of 185 and 50 (63%) of 80. CONCLUSIONS: Retinal reattachment was achieved in the majority of eyes using vitrectomy and silicone oil retinal tamponade. Complication rates generally were less frequent in CMV eyes, but follow-up was shorter in this group of patients, largely because of reduced life expectancy. Cataract frequently developed in phakic eyes of study patients. Use of 1000-centistoke silicone oil can be considered in the management of complex retinal detachments associated with multiple etiologies.

Calorimetric analyses of the interaction between SecB and its ligands.

SecB is a chaperone in Escherichia coli dedicated to export of proteins from the cytoplasm to the periplasm and outer membrane. It functions to bind and deliver precursors of exported proteins to the translocation apparatus before they fold into their native structures, thus maintaining them in a competent state for translocation across the membrane. The natural ligands of SecB are precursor proteins containing leader sequences. There are numerous reports in the literature indicating that SecB does not specifically recognize the leader peptides. However, two published investigations have concluded that the leader peptide is the recognition element (Watanabe M, Blobel G. 1989. Cell 58:685-705; Watanabe M, Blobel G. 1995. Proc Natl Acad Sci USA 92:10133-10136). In this work we use titration calorimetry to show that SecB binds two physiological ligands, which contain leader sequences, with no higher affinity than the same molecules lacking their leader sequences. Indeed, for one ligand the presence of the leader sequence reduces the affinity. Therefore, it can be concluded that the leader sequence provides no positive contribution to the binding energy.

Chaperone SecB from Escherichia coli mediates kinetic partitioning via a dynamic equilibrium with its ligands.

We have shown that the complexes between SecB, a chaperone from Escherichia coli, and two physiological ligands, galactose-binding protein and maltose-binding protein, are in rapid, dynamic equilibrium between the bound and free states. Binding to SecB is readily reversible, and each time the ligand is released it undergoes a kinetic partitioning between folding to its native state and re-binding to SecB. Binding requires that the polypeptide be devoid of tertiary structure; once the protein has folded, it is no longer a ligand. Conditions were established in which folding of the polypeptides was sufficiently slow so that at each cycle of dissociation rebinding was favored over folding and a kinetically stable complex between SecB and each polypeptide ligand was observed. Evidence that the ligand is continually released to the bulk solution and rebound was obtained by altering the conditions to increase the rate of folding of each ligand so that folding of the ligand was faster than reassociation with SecB thereby allowing the system to partition to free SecB and folded polypeptide ligand. We conclude that complexes between the chaperone SecB and ligands are in dynamic, rapid equilibrium with the free states. This mode of binding is simpler than that documented for chaperones that function to facilitate folding such as the Hsp70s and Hsp60s, where hydrolysis of ATP is coupled to the binding and release of ligands. This difference may reflect the fact that SecB does not mediate folding but is specialized to facilitate protein export. Without a requirement for exogenous energy it efficiently performs its sole duty: to keep proteins in a nonnative conformation and thus competent for export.

Blebitis, early endophthalmitis, and late endophthalmitis after glaucoma-filtering surgery.

PURPOSE: The differentiating characteristics in blebitis and early and late endophthalmitis after glaucoma filtration surgery are reviewed. METHODS: All admission records and operative reports, as well as available office notes, on patients with blebitis or bleb-associated endophthalmitis admitted to a large referral eye center from 1985 to 1995 were reviewed retrospectively. RESULTS: Ten cases of blebitis and 33 cases of bleb-associated endophthalmitis were identified. One patient with blebitis progressed to culture-positive endophthalmitis. Of the 33 cases of bleb-associated endophthalmitis, there were 6 cases of early endophthalmitis (before postoperative week 6) and 27 cases of late endophthalmitis. In early endophthalmitis, Staphylococcus epidermidis was isolated on vitreous culture in 4 (67%) of 6 cases, whereas in late endophthalmitis, this organism was isolated in only 1 (4%) of 27 cases. In the 27 late cases, Streptococcus species and gram-negative organisms comprised 48% of isolates; of 33 cases of endophthalmitis, 15 (45%) demonstrated no growth on vitreous culture. Patients with endophthalmitis fared more poorly than those with blebitis in terms of visual outcome. CONCLUSIONS: Because blebitis may be prodromal to endophthalmitis, aggressive antimicrobial therapy, perhaps with oral quinolones, is warranted. In addition, patients with blebitis should be observed closely to identify extension into the vitreous cavity so that intravitreous antibiotics can be administered in a timely fashion. Finally, clinicians should not extrapolate the results of the Endophthalmitis Vitrectomy Study to the postfiltration surgery endophthalmitis given the differing pathogenesis and unique spectrum of organisms.

Uveal lymphoid neoplasia: a clinical-pathologic correlation and review of the early form.

We report three cases of uveal lymphoid neoplasia that were diagnosed early in their course. One case exhibited a posterior form, presenting with progressive hyperopia from a serous-macular detachment and choroidal involvement along with retrobulbar involvement. This patient was treated with proton beam irradiation. Two cases displayed an anterior form, with fixed fleshy epibulbar masses resembling salmon patches, and choroidal involvement. The histologic findings from biopsy of these anterior masses are presented. One of these patients was treated with complete excision of the mass and double freeze-thaw cryotherapy of the scleral bed, and the other patient was treated with conventional photon beam irradiation. The clinical features of uveal lymphoid neoplasia in its early form are discussed. Evaluation and treatment strategies for these early forms of uveal lymphoid neoplasia are reviewed.

Binding of SecB to ribosome-bound polypeptides has the same characteristics as binding to full-length, denatured proteins.

The interaction of the chaperone SecB with ribosome-bound polypeptides that are in the process of elongation has been studied using an in vitro protein synthesis system. The binding is characterized by the same properties as those demonstrated for the binding of SecB to full-length proteins that are in nonnative conformation: it is readily reversible and has no specificity for the leader peptide. In addition, it is shown that the growing polypeptide chains must achieve a critical length to bind tightly enough to allow their isolation in complex with SecB. This explains the longstanding observation that, even when export is cotranslational, it begins late in synthesis. Furthermore, the required length is approximately the same as the length that defines the binding frame within denatured, full-length proteins bound to SecB.

Posterior segment complications after vitrectomy for macular hole.

PURPOSE: The purpose of this study is to assess the rate of posterior segment complications after vitreous surgery for macular holes and to evaluate the effect of such complications on final visual outcome. METHODS: The authors reviewed retrospectively all cases of vitreous surgery for macular holes performed between June 1990 and October 1993. Among 98 patients with a followup of 3 months or more, all patients with posterior segment complications during the postoperative course were identified. The rate of complications was compared with that seen after vitreous surgery for macular pucker performed by the same surgeons. RESULTS: Posterior segment complications were noted in 23 (23%) of 98 patients. These included peripheral retinal breaks (3%), rhegmatogenous retinal detachment from a peripheral retinal break (14%), enlargement of the hole (2%), late reopening of the hole (2%), retinal pigment epithelium loss under the hole (1%), photic toxicity (1%), and endophthalmitis (1%). In 40% of these eyes, the final visual acuity was two lines or more below preoperative visual acuity. When compared with the macular pucker group, the rate of posterior segment complications, in particular the rate of peripheral retinal tears and detachments, was significantly higher (P < or = 0.05). CONCLUSIONS: The authors conclude that visually significant posterior segment complications may occur after vitrectomy for macular hole, and the rate of these complications appears to be higher than expected.

Determination of the binding frame within a physiological ligand for the chaperone SecB.

The hallmark of the class of proteins called chaperones is the amazing ability to bind tightly to a wide array of polypeptide ligands that have no consensus in sequence; chaperones recognize non-native structure. As a step in the elucidation of the molecular mechanism of such remarkable binding, we have characterized complexes between the bacterial chaperone SecB and a series of ligands related to maltose-binding protein. SecB interacts at multiple sites on its polypeptide ligand. The entire binding region covers approximately half of the primary sequence of maltose-binding protein and comprises contiguous sites positioned around the center of the sequence.