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Receptors internalized by endocytosis can return to the plasma membrane (PM) directly from early endosomes (EE; fast recycling) or they can traffic from EE to the endocytic recycling compartment (ERC) and recycle from there (slow recycling). How receptors are sorted for trafficking along these two pathways remains unclear. Here we show that autosomal recessive hypercholesterolemia (ARH) is required for trafficking of megalin, a member of the LDL receptor family, from EE to the ERC by coupling it to dynein; in the absence of ARH, megalin returns directly to the PM from EE via the connecdenn2/Rab35 fast recycling pathway. Binding of ARH to the endocytic adaptor AP-2 prevents fast recycling of megalin. ARH-mediated trafficking of megalin to the ERC is necessary for γ-secretase mediated cleavage of megalin and release of a tail fragment that mediates transcriptional repression. These results identify a novel mechanism for sorting receptors for trafficking to the ERC and link ERC trafficking to regulated intramembrane proteolysis (RIP) and expression of megalin.

Purpose: To review results of traumatic cataracts in children., Methods: This is a retrospective cohort study done at a tertiary eye care center at the junction of Gujarat, Madhya Pradesh, and Rajasthan states in central western India. We enrolled children with specific inclusion criteria, examined their eyes to review the comorbidities due to trauma, performed surgery for traumatic cataracts, and implanted a lens, treating amblyopia if applicable. The patients were re-examined 6 weeks postoperatively. We divided the traumatic cataract cases into open-globe (group 1) and closed-globe (group 2) groups according to the ocular trauma based on the Birmingham Eye Trauma Terminology System and compared the determinants of visual acuity., Result: Our cohort of 354 eyes with traumatic cataracts in children included 287 eyes in group 1 and 67 in group 2. Six weeks postoperatively, the visual acuity in the operated eye was >20/200 in 181 (63%) and =20/40 in 109 (38%) eyes in the open-globe group and >20/200 in 36 (53%) and =20/40 in 16 (22.4%) eyes in the closed-globe group (p = 0.143), and the difference between the groups was not significant in children. Overall, 125 (35.3%) eyes gained a visual acuity at 6 weeks of =20/40 and >20/200 in 214 (61.3%) cases., Conclusions: Satisfactory visual outcome can be achieved in children with traumatic cataracts, with no significant difference found among open- and closed-globe injuries in the pediatric age group.

Objective: To validate the predictive value of the Ocular Trauma Score (OTS) in injury cases with traumatic cataracts., Design: Retrospective cohort study., Participants: A total of 787 eyes., Methods: A total of 787 eyes of 787 subjects with traumatic cataracts were enrolled using specific inclusion criteria. The eyes were examined to review comorbidities caused by trauma. Surgery was performed for traumatic cataracts, lenses were implanted, and patients were treated for amblyopia, as applicable. The patients were reexamined 6 weeks postoperatively. On the basis of ocular trauma described according to the Birmingham Eye Trauma Terminology System, the patients were divided into 2 traumatic cataract groups: open globe injury and closed globe injury. The relationship of visual acuity (VA) with demographic and clinical variables was analyzed. The visual outcomes were predicted using the OTS, and the predictions were compared with the actual outcomes using statistical tests., Main Outcome Measures: Visual acuity., Results: At 6 weeks postoperatively, 245 eyes (31%) had a VA ≥ 20/40 and 480 eyes (61.0%) had a VA >20/200. The OTS prediction was not significantly different when compared with actual visual outcome at 6 weeks postoperatively in all OTS categories., Conclusions: The relationship of VA at 6 weeks with demographic and clinical variables was analyzed. In this study, the OTS was found as a reliable tool to predict visual outcome in cases of traumatic cataracts 6 weeks postoperatively., (Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

A case of 18F-FDG PET in pulmonary alveolar microlithiasis with bilateral reticulonodular opacities in the middle and lower zones on routine chest roentgenogram and calcification in the peribronchovascular interstitium,peribronchovascular and subpleural intralobular septal regions, and visceral pleura on high-resolution chest CT is presented. 18F-fluoride PET demonstrated intense tracer uptake in the lower zone of both lungs corresponding to CT findings. Minimal FDG activity in areas of dense calcification suggests minimal or no inflammation. This could account for the absence of symptoms in most cases despite remarkable imaging findings.

Purpose: To validate the predictive value of the Ocular Trauma Score (OTS) in children with traumatic cataract., Design: Retrospective cohort study., Setting: Tertiary eye care center at nexus of Gujarat, Madhya Pradesh, and Rajasthan states, central Western India., Methods: After meeting inclusion criteria, eyes with traumatic cataract were enrolled and examined to review comorbidities caused by trauma. Surgery was performed for traumatic cataract, intraocular lenses were implanted, and patients were treated for amblyopia, as applicable. Patients were reexamined 6 weeks postoperatively. Based on ocular trauma described according to the Birmingham Eye Trauma Terminology System, the patients were divided into 2 traumatic cataract groups: open-globe injury and closed-globe injury. The relationship between final corrected distance visual acuity (CDVA) and demographic and clinical variables was analyzed. Visual outcomes were predicted using the OTS, and the predictions were compared with actual outcomes using statistical tests., Results: The study enrolled 354 children. Six weeks postoperatively, the CDVA was better than 20/200 in 181 eyes (63.0%) and 20/40 or better in 110 eyes (38.4%) in the open-globe group and better than 20/200 in 36 eyes (53%) and 20/40 or better in 15 eyes (22.4%) in the closed-globe group. The differences between the groups were not significant (P=.143). Of all eyes, 214 (61.3%) achieved a final CDVA of better than 20/200 and 123 eyes (35.3%), of 20/40 or better., Conclusion: The OTS was a reliable predictor of the final visual outcome in cases of pediatric traumatic cataract., (Copyright © 2012 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Hypercholesterolemia, high serum cholesterol in the form of LDL, is a major risk factor for atherosclerosis. LDL is mostly degraded in the liver after its cellular internalization with the LDL receptor (LDLR). This clathrin-mediated endocytosis depends on the protein autosomal recessive hypercholesterolemia (ARH), which binds the LDLR cytoplasmic tail. Mutations in either the LDLR tail or in ARH lead to hypercholesterolemia and premature atherosclerosis. Despite the significance of this interaction for cholesterol homeostasis, no structure of either ARH or the LDLR tail is available to determine its molecular basis. We report the crystal structure at 1.37-Å resolution of the phosphotyrosine-binding (PTB) domain of ARH in complex with an LDLR tail peptide containing the FxNPxY(0) internalization signal. Surprisingly, ARH interacts with a longer portion of the tail than previously recognized, which extends to I(-7)xF(-5)xNPxY(0)QK(+2). The LDLR tail assumes a unique "Hook"-like structure with a double β-turn conformation, which is accommodated in distinctive ARH structural determinants (i.e., an extended backbone hydrogen-bonding platform, three hydrophobic helical grooves, and a hydrophobic pocket for Y(0)). This unique complementarity differs significantly in related PTB proteins and may account for the unique physiological role of these partners in the hepatic uptake of cholesterol LDL. Moreover, the unusual hydrophobic pocket for Y(0) explains the distinctive ability of ARH to internalize proteins containing either FxNPxY(0) or FxNPxF(0) sequences. Biophysical measurements reveal how mutations associated with hypercholesterolemia destabilize ARH and its complex with LDLR and illuminate LDL internalization defects seen in patients.

Accidental ingestion of phenytoin can lead to severe neurological sequelae. Charcoal hemoperfusion decreases phenytoin levels but has never been reported before in children. We present a child with accidental ingestion of phenytoin who responded to charcoal hemoperfusion.

Objective: The objective of this work is to compare final visual outcomes in cases of surgically treated traumatic cataract between open-globe and closed-globe groups, as classified by the Birmingham Eye Trauma Terminology system., Design: Observational cohort study., Setting: Tertiary eye-care center at the trijunction of Gujarat, Madhya Pradesh, and Rajasthan states in central western India., Methods: We enrolled patients meeting specific inclusion criteria, examined their eyes to review any co-morbidities due to trauma, performed surgery for traumatic cataracts, and implanted lenses. The patients were re-examined 6 weeks postoperatively. We classified the cases of traumatic cataract as either open-globe (group 1) or closed-globe (group 2), according to the Birmingham Eye Trauma Terminology (BETT) system, and compared visual acuity., Outcome Measures: Visual Acuity., Results: Our cohort of 687 eyes with traumatic cataracts included 496 eyes in group 1 and 191 in group 2. Six weeks postoperatively, the visual acuity was >20/60 in 298 (58%) and 75 (39.1%) operated eyes in groups 1 and 2, respectively (p < 0.001, ANOVA). At follow-up, >20/60 vision was significantly higher in group 1 than in group 2 (OR = 1.61; 95% CI, 0.85-3.02). Overall, 373 eyes (54.3%) regained final visual acuity >20/60., Conclusions: Open-globe injury has a more favorable prognosis for satisfactory (>20/60) visual recovery after management of traumatic cataracts.

Purpose: There are no clear guidelines to treat traumatic cataract. This study was conducted to provide evidence-based care to patients with traumatic cataracts and to examine the effect of the time interval between injury and the first intervention on the final visual outcome., Methods: In a prospective cohort study, all patients presenting to our hospital with traumatic cataracts between January 2003 and December 2009 were enrolled. Information regarding demographics and ocular trauma was collected on the pretested World Eye Trauma Registry form for both the first and follow-up visits. In particular, we collected specific information on the time interval between the injury and intervention. The relationship between this time interval and the final visual outcome was analyzed. The study was conducted at a tertiary eye care center, in Dahod, at the junction of Gujarat, Madhya Pradesh, and Rajasthan states, in central western India., Results: The time interval between the injury and first intervention had a significant effect on the final visual outcome (p = 0.02, chi2 test)., Conclusions: The morphology of traumatic cataracts plays an important role in determining the appropriate surgical technique and the final visual outcome.

Objective: To investigate the outcome of secondary intraocular lens implantation, compare final visual outcome between different categories of surgeon, and evaluate care provided by teaching hospitals to patients with capsular complications., Setting: Teaching hospital., Design: Retrospective study. Subjects were recruited by examination of electronic medical records. All patients operated for corrective surgery following capsular complications during cataract surgery were included. All patient medical records were reviewed, and data were collected for 359 eyes., Main Outcome Measures: Visual acuity and major complications. All collected data were entered into Microsoft Excel and analyzed by SPSS 17 software using cross tabulation and Chi-squared tests., Results: Surgical intervention made a significant difference to the final visual outcome (P < 0.001). The category of the trainee had a significant effect on the final visual outcome (P = 0.021)., Conclusion: Capsular complications during cataract surgery should be surgically treated to improve outcome.

Aim To study the morphology of traumatic cataract as an important predictor for final visual outcome after treatment of traumatic cataracts. Setting Tertiary eye care centre in Dahod at the trijunction of Gujarat, Madhya Pradesh, and Rajasthan states in central western India. Methods This was a prospective observational cohort study among all patients presenting at the hospital with traumatic cataracts between January 2003 and December 2009. All information regarding demographic and ocular trauma was collected on a pretested World Eye Trauma Registry form for both the first visit and follow-up. In particular, the authors collected specific information about the morphology of traumatic cataracts; the surgical technique was determined accordingly. Data were entered and analysed with regard to the relationship between type of trauma and resulting injury, results achieved with particular surgical techniques, and the relationship between morphology and final visual outcome. Outcome measures Final visual outcome. Results Traumatic cataracts of different morphologies showed significant differences in the final visual outcome (χ(2) test, p=0.014). Conclusion The morphology of traumatic cataract plays an important role in the final visual outcome.

Aim: The aim was to evaluate the visual recovery after managing traumatic cataracts and determine the predictors of a better visual prognosis., Materials and Methods: This was a prospective study. We enrolled patients with specific inclusion criteria, examined their eyes to review the comorbidities due to trauma, performed surgery for traumatic cataracts, and implanted lenses. The patients were reexamined 6 weeks postoperatively. We divided the cases of traumatic cataract into two groups, the "open globe" (Group 1) and "closed globe" (Group 2) groups, according to the ocular trauma based on the Birmingham Eye Trauma Terminology System (BETTS) and compared the determinants of visual acuity., Results: Our cohort of 555 eyes with traumatic cataracts included 394 eyes in Group 1 and 161 in Group 2. Six weeks postoperatively, the visual acuity in the operated eye was >20/60 in 193 (48%) and 49 (29%) eyes in Groups 1 and 2, respectively (P = 0.002, ANOVA). At follow-up, >20/60 vision was significantly higher in Group 1 than in Group 2 (OR = 1.61; 95% CI, 0.85-3.02). Overall 242 (43.5%) eyes gained a final visual acuity of >20/60., Conclusions: Open globe injury has a favorable prognosis for satisfactory (>20/60) visual recovery after the management of traumatic cataracts.

Background/aims: Mutations in the inwardly-rectifying K+ channel KCNJ10/Kir4.1 cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (EAST syndrome). KCNJ10 is expressed in the kidney distal convoluted tubule, cochlear stria vascularis and brain glial cells. Patients clinically diagnosed with EAST syndrome were genotyped to identify and study mutations in KCNJ10., Methods: Patient DNA was sequenced and new mutations identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K+ currents were measured by two-electrode voltage clamping., Results: Three new mutations in KCNJ10 (p.R65C, p.F75L and p.V259fs259X) were identified, and mutation p.R297C, previously only seen in a compound heterozygous patient, was found in a homozygous state. Wild-type human KCNJ10-expressing oocytes showed strongly inwardly-rectified currents, which by comparison were significantly reduced in all the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba2+ demonstrated residual function in all mutant channels (p < 0.05) but V259X., Conclusion: This study confirms that EAST syndrome can be caused by many different mutations in KCNJ10 that significantly reduce K+ conductance. EAST syndrome should be considered in any patient with a renal Gitelman-like phenotype with additional neurological signs and symptoms like ataxia, epilepsy or sensorineural deafness., (Copyright © 2011 S. Karger AG, Basel.)

Injuries to the articular cartilage of the knee are common. They alter the normal distribution of weightbearing forces and predispose patients to the development of degenerative joint disease. The management of focal chondral lesions continues to be problematic for the treating orthopaedic surgeon. Although many treatment options are currently available, none fulfill the criteria for an ideal repair solution: a hyaline repair tissue that completely fills the defect and integrates well with the surrounding normal cartilage. Autologous chondrocyte implantation (ACI) is a relatively new cell-based treatment method for full-thickness cartilage injuries that in recent years has increased in popularity, with early studies showing promising results. The current article reviews the nature of cartilage lesions in the knee and the treatment modalities utilized in their management, focusing on the role ACI plays in the surgical treatment of these complex injuries.

An 8-year-old girl presented with complaints of bilateral epiphora since birth. The patient had congenital punctal and canalicular atresia combined with congenital fistula. She was treated successfully with surgery. A review of the literature indicated very few reports of surgical treatment of such cases.

Femoroacetabular impingement results from an abnormal contact between the femur and the pelvis. This abnormal contact leads to developmental changes in the femoral neck, labrum, and acetabulum. Secondary to the altered hip joint mechanics, chondral damage occurs and initiates the degenerative process, eventually leading to osteoarthritis. Numerous etiologies have been implicated in femoroacetabular impingement, and a variety of treatment algorithms have been established, with no definitive gold standard. However, the treatment of this disorder with joint preserving techniques offers a viable option between the extremes of nonoperative treatment and total joint arthroplasty.

Numerous proteins involved in endocytosis at the plasma membrane have been shown to be present at novel intracellular locations and to have previously unrecognized functions. ARH (autosomal recessive hypercholesterolemia) is an endocytic clathrin-associated adaptor protein that sorts members of the LDL receptor superfamily (LDLR, megalin, LRP). We report here that ARH also associates with centrosomes in several cell types. ARH interacts with centrosomal (gamma-tubulin and GPC2 and GPC3) and motor (dynein heavy and intermediate chains) proteins. ARH cofractionates with gamma-tubulin on isolated centrosomes, and gamma-tubulin and ARH interact on isolated membrane vesicles. During mitosis, ARH sequentially localizes to the nuclear membrane, kinetochores, spindle poles and the midbody. Arh(-/-) embryonic fibroblasts (MEFs) show smaller or absent centrosomes suggesting ARH plays a role in centrosome assembly. Rat-1 fibroblasts depleted of ARH by siRNA and Arh(-/-) MEFs exhibit a slower rate of growth and prolonged cytokinesis. Taken together the data suggest that the defects in centrosome assembly in ARH depleted cells may give rise to cell cycle and mitotic/cytokinesis defects. We propose that ARH participates in centrosomal and mitotic dynamics by interacting with centrosomal proteins. Whether the centrosomal and mitotic functions of ARH are related to its endocytic role remains to be established.

Justification: In 2001, the Indian Pediatric Nephrology Group formulated guidelines for management of patients with steroid sensitive nephrotic syndrome. In view of emerging scientific evidence, it was felt necessary to review the existing recommendations., Process: Following a preliminary meeting in March 2007, a draft statement was prepared and circulated among pediatric nephrologists in the country to arrive at a consensus on the evaluation and management of these patients., Objectives: To revise and formulate recommendations for management of steroid sensitive nephrotic syndrome., Recommendations: The need for adequate cortico-steroid therapy at the initial episode is emphasized. Guidelines regarding the initial evaluation, indications for renal biopsy and referral to a pediatric nephrologist are updated. It is proposed that patients with frequently relapsing nephrotic syndrome should, at the first instance, be treated with long-term, alternate-day prednisolone. The indications for use of alternative immunosuppressive agents, including levamisole, cyclophosphamide, mycophenolate mofetil and cyclosporin are outlined. The principles of dietary therapy, management of edema, and prevention and management of complications related to nephrotic syndrome are described. These guidelines, formulated on basis of current best practice, are aimed to familiarize physicians regarding management of children with steroid sensitive nephrotic syndrome.

Lung vascular lesions in pulmonary arterial hypertension (PAH) are characterized by enlarged, vacuolated ("megalocytotic") pulmonary arterial endothelial (PAEC) and smooth muscle cells (PASMC). We have recently proposed that dysfunction of vesicle tethers, soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs), and SNAP receptors (SNAREs), leading to disruptions of intracellular trafficking in the Golgi to plasma membrane (centrifugal) and the plasma membrane to cell interior (centripetal) directions is a key causal mechanism in this disease. In PAH, there was a reciprocal relationship between loss of caveolin-1 (cav-1) in PAECs and increased expression of "activated" tyrosine-phosphorylated STAT3 (PY-STAT3) associated with a block in centrifugal trafficking to/through the Golgi organelle. In the present study, we investigated 1) whether centripetal trafficking of STAT3 and PY-STAT3 in PAECs and PASMCs was membrane-associated, and 2) whether this might be affected in PAH. Immunofluorescence and live cell imaging studies showed that, in both PAEC and PASMC, STAT3 was associated with cytoplasmic vesicles partially colocalizing with markers of the endolysosomal compartments (clathrin, EEA1, Rab5, Rab11, and LAMP1). Overexpression of cav-1 increased the targeting of STAT3 to lysosomes and inhibited STAT3 transcriptional activity. Exposure of PAECs to monocrotaline (MCT) pyrrole, which causes PAH in the rat, led to a loss of caveolar STAT3 with increased sequestration of STAT3 and PY-STAT3 in endosomes. In vivo, marked cytoplasmic sequestration of activated PY-STAT3 was a common feature in PAEC in the rat/MCT model and in cells in the proliferative arterial and plexiform lesions in PAH in humans. These data highlight the epigenetic regulation of centripetal cytokine and growth-factor signaling pathways and its modulation in PAH.

Background: The authors present the epidemiology of ocular trauma and visual status before and after their management from 2003 to 2005 at a hospital in a tribal area of India., Methods: This was a historical cohort study. Persons exposed to open and closed globe types of injuries were reviewed. Ophthalmologists examined patients with the help of bio-microscope and also noted their visual acuity. Ocular Injuries were classified according to Birmingham Eye Trauma Terminology System (BETTS). Epidemiological profile, causes of injury, ocular status and visual gain after management were noted. Percentage scattergrams were plotted for both groups of injuries to evaluate visual gains. Binary logistic regression analysis was carried out to determine the predictors of visual outcomes following ocular trauma management., Results: Of the 2,607 persons with ocular trauma, 'open globe' and 'closed globe' types of injuries were found in 451 and 2,156 persons respectively. Within 24 hours of injury, only 1,355 patients (52%) had consulted ophthalmologists. Improvement in vision following management was noted in 133 persons (29.4%) with 'open globe' injuries and 217 persons (48.1%) with 'closed globe' injuries. In 845 patients (39.5%), vision could be retained at same level after management., Conclusions: Agents and activities responsible for ocular injuries in our study differed from those reported in the industrialized countries. Late presentation was very common in our tribal area. Management improved the vision in eyes with trauma, but eyes with 'closed globe' type of ocular injuries had better results than 'open globe' type.

Purpose: There are many options for arthroscopic knots including the type of knot and suture material used. The current investigation evaluated knot properties using 3 high-strength suture materials tied in 5 common arthroscopic knot configurations., Methods: Four arthroscopic sliding knots including the Roeder, Weston, SMC, and Tennessee Slider and an arthroscopic nonsliding Surgeon's knot were evaluated. Each knot was tied with each of 3 No. 2 polyblended suture types (Fiberwire [Arthrex, Naples, FL], Ultrabraid [Smith & Nephew, Andover, MA], and Orthocord [Mitek, Raynham, MA]). Each configuration was tied 8 times, for a total of 120 samples. Loop security and knot security were then evaluated by using a previously described protocol comparing the different knot types and suture material., Results: With respect to loop security, Orthocord performed better than the other tested suture materials, producing on average smaller knot loops. For the nonsliding Surgeon's knot, there was no difference in loop security observed between suture types. For the Roeder knot, Fiberwire had superior knot security compared with Ultrabraid and Orthocord (P < .001). For the Weston knot, Ultrabraid showed superior knot security compared with Orthocord (P < .02). Knot security for the Tenessee slider knot was better for both Fiberwire and Ultrabraid compared with Orthocord (P < .001, respectively). Similar results were seen with the SMC knot, with Fiberwire and Ultrabraid outperforming Orthocord (P < .001, respectively). The nonsliding Surgeon's knot had significantly lower mean loads to failure compared with arthroscopic sliding knots for each tested suture material (P < .02 for all comparisons)., Conclusions: Loop security and knot security varied depending on the type of knot tied and suture material used. Arthroscopic sliding knots performed better than the nonsliding Surgeon's knot., Clinical Relevance: Surgeons should try to use sliding knots instead of Surgeon's knots when using polyblend suture material. Differences between the brands in this suture category will change the characteristics of the knots thrown and may ultimately affect tissue fixation.

Monocrotaline (MCT)-induced pulmonary hypertension (PH) in the rat is a widely used experimental model. We have previously shown that MCT pyrrole (MCTP) produces loss of caveolin-1 (cav-1) and endothelial nitric oxide synthase from plasma membrane raft microdomains in pulmonary arterial endothelial cells (PAEC) with the trapping of these proteins in the Golgi organelle (the Golgi blockade hypothesis). In the present study, we investigated the mechanisms underlying this intracellular trafficking block in experiments in cell culture and in the MCT-treated rat. In cell culture, PAEC showed trapping of cav-1 in Golgi membranes as early as 6 h after exposure to MCTP. Phenotypic megalocytosis and a reduction in anterograde trafficking (assayed in terms of the secretion of horseradish peroxidase derived from exogenously transfected expression constructs) were evident within 12 h after MCTP. Cell fractionation and immunofluorescence techniques revealed the marked accumulation of diverse Golgi tethers, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), and soluble NSF attachment proteins (SNAPs), which mediate membrane fusion during vesicular trafficking (GM130, p115, giantin, golgin 84, clathrin heavy chain, syntaxin-4, -6, Vti1a, Vti1b, GS15, GS27, GS28, SNAP23, and alpha-SNAP) in the enlarged/circumnuclear Golgi in MCTP-treated PAEC and A549 lung epithelial cells. Moreover, NSF, an ATPase required for the "disassembly" of SNARE complexes subsequent to membrane fusion, was increasingly sequestered in non-Golgi membranes. Immunofluorescence studies of lung tissue from MCT-treated rats confirmed enlargement of perinuclear Golgi elements in lung arterial endothelial and parenchymal cells as early as 4 days after MCT. Thus MCT-induced PH represents a disease state characterized by dysfunction of Golgi tethers, SNAREs, and SNAPs and of intracellular vesicular trafficking.

Raft and caveolar microdomains have been proposed to participate in numerous cellular functions including signal transduction, cholesterol trafficking, and vesicular sorting. Traditional methods of isolation of rafts from cultured cells and tissue samples have exploited the biochemical properties of these microdomains, i.e., their relative resistance to solubilization by nonionic detergents (at 4 degrees C) and their light buoyant density attributable to their high content of cholesterol and sphingolipids. Thus, a common way to isolate raft microdomains has been their separation on a density gradient in the presence of 0.5-1% Triton X-100 (Bochringer Mannheim Roche Applied Sciences Indianapolis, IN or Sigma-Aldrich, St. Louis, MO). This and other detergent-based methods have been discussed. However, the use of detergents may not be favorable because of artifacts that may arise with their use. (The possibility of rafts solely as detergent-induced artifacts appears to have been diffused by a number of biochemical and biophysical studies that strongly demonstrate the presence of a liquid-ordered phase within biological membranes.) In this chapter, three methods are reviewed to isolate rafts from cultured cells without the use of detergents. Two of these, the sodium carbonate and OptiPrep (Sigma-Aldrich St. Louis, MO) methods, are based on gradient separation and can be used to isolate rafts in general, whereas the third is a magnetic-bead immunoisolation approach and might be used to isolate subpopulations of rafts enriched for different markers such as caveolin-1, flotillin (reggie proteins), or other suitable markers. Together these methods allow for a detergent-free isolation of rafts for biochemical, proteomic, and microscopic studies.

Articular cartilage defects are common and play a significant role in degenerative joint disease. Cartilage is unable to regenerate, secondary to an inherent lack of vascular supply, thus, various techniques have been described in an attempt to treat and potentially restore these defects. Treatment decisions should be based on appropriate evaluation and classification of the pathology. Only then can the surgeon choose to perform a repair or a restoration of the articular surface. Current literature and techniques for the treatment of articular cartilage defects are reviewed, with an algorithm developed for the management of articular cartilage defects by orthopaedic surgeons.

Mechanisms to maintain blood pressure in the face of infection are critical to survival. The angiotensinogen (AGT) gene locus is an important component of this response. Thus the AGT gene, expressed predominantly by liver cells, is known to be a positive acute phase reactant. We have previously demonstrated activation of the AGT promoter in hepatocytes through the IL6/STAT3 signaling mechanism. We have now investigated whether IFN-gamma, a cytokine also induced in response to diverse infections, can regulate AGT gene expression, and have elucidated the molecular mechanism involved. IFN gamma treatment up-regulated AGT mRNA level and promoter activity in Hep3B hepatocytes. Sequential deletion of the promoter from the 5' side suggested the major IFN gamma responsive DNA element to be between -303 and -103. This region contained a candidate STAT1-binding site between -271 and -279. EMSA and chromatin immuno-precipitation (ChIP) assays confirmed that IFN-gamma treatment induced the binding of STAT1 to this element. Reporter constructs containing this AGT promoter derived element in a multimerized context but not a mutant version were responsive to IFN gamma. Moreover mutating this STAT1 element in the context of the wild-type AGT holo promoter reduced responsiveness to IFN gamma. In contrast to the clear synergism between dexamethasone and IL 6 in the upregulation of the AGT promoter (through interaction between GR and STAT3), the combination of IFN gamma with IL 6 or with dexamethasone did not further increase AGT promoter activity suggesting that the IFN gamma/STAT1 pathway represents a separate signaling mechanism. These data highlight the redundancy in cytokine-mediated host response pathways aimed at the maintenance of blood pressure during infection.

Signal transduction from the plasma membrane to the nucleus by STAT proteins is widely represented as exclusively a soluble cytosolic process. Using cell-fractionation methods, we observed that approximately 5% of cytoplasmic STAT3 was constitutively associated with the purified early endosome (EE) fraction in human Hep3B liver cells. By 15-30 min after interleukin-6 (IL-6) treatment, up to two-thirds of cytoplasmic Tyr-phosphorylated STAT3 can be associated with the purified early endosome fraction (Rab-5-, EEA1-, transferrin receptor-, and clathrin-positive fraction). Electron microscopy, immunofluorescence, and detergent dissection approaches confirmed the association of STAT3 and PY-STAT3 with early endosomes. STAT3 was constitutively associated with clathrin heavy chain in membrane and in the 1- to 2-MDa cytosolic complexes. The membrane association was dynamic in that, within 15 min of treatment with the vicinal-thiol cross-linker phenylarsine oxide, there was a dramatic increase in bulk STAT3 association with sedimentable membranes. The functional contribution of PY-STAT3 association with the endocytic pathway was evaluated in transient transfection assays using IL-6-inducible STAT3-reporter-luciferase constructs and selective regulators of this pathway. STAT3-transcriptional activation was inhibited by expression constructs for dominant negative dynamin K44A, epsin 2a, amphiphysin A1, and clathrin light chain but enhanced by that for the active dynamin species MxA. Taken together, these studies emphasize the contribution of the endocytic pathway to productive IL-6/STAT3 signaling.

The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a "megalocytosis" phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the "Golgi blockade" hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and alpha-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1alpha and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis.

Pyrrolizidine alkaloids initiate disease in the lung (pulmonary hypertension), liver (veno-occlusive disease and cirrhosis), and kidneys (afferent arteriolar block and mesangiolysis) by inducing a megalocytotic phenotype in target endothelial and parenchymal cells. A "hit-and-run" type of exposure to the bioactive pyrrolizidine results, within 2-3 days, in enlarged cells with large nuclei and enlarged Golgi and endoplasmic reticulum, while the cells remain in G2/M block. In the present study, we recapitulated monocrotaline pyrrole (MCTP)-induced megalocytosis in cultures of bovine pulmonary arterial endothelial cells (PAEC), human Hep3B hepatocytes, human type II-like alveolar epithelial cells (A549), and human pulmonary arterial smooth muscle cells (PASMC) and investigated the subcellular mechanism involved. There was an inverse relationship between reduction in caveolin (Cav)-1 levels and stimulation of promitogenic STAT3 and ERK1/2 cell signaling. In megalocytotic PAEC, the Golgi scaffolding protein GM130 was shifted from membranes with heavy density to those with a lighter density. This lighter Golgi fraction was enriched for hypo-oligomeric Cav-1, indicating dysfunctional trafficking of cargo. Immunofluorescence imaging studies confirmed the trapping of Cav-1 in a GM130-positive Golgi compartment. There was an increase in Ser25 phosphorylation of GM130 (typically a prelude to Golgi fragmentation and mitosis) and increased association between pGM130, cdc2 kinase, and Cav-1. Nevertheless, megalocytotic MCTP-treated cells showed reduced entry into mitosis upon stimulation with 2-methoxyestradiol (2-ME), reduced 2-ME-induced Golgi fragmentation, and a slowing of Golgi reassembly after nocodazole-induced fragmentation. These data suggest that a disruption of the trafficking and mitosis sensor functions of the Golgi may represent the subcellular mechanism leading to MCTP-induced megalocytosis ("the Golgi blockade hypothesis").

Background: In the monocrotaline (MCT)-treated rat, there is marked stimulation of DNA synthesis and megalocytosis of pulmonary arterial endothelial cells (PAECs) within 3 to 4 days, followed by pulmonary hypertension (PH) 10 to 14 days later. Growing evidence implicates caveolin-1 (cav-1) in plasma membrane rafts as a negative regulator of promitogenic signaling. We have investigated the integrity and function of endothelial cell-selective cav-1alpha/raft signaling in MCT-induced PH., Methods and Results: Although PH and right ventricular hypertrophy developed by 2 weeks after MCT, a reduction in cav-1alpha levels in the lung was apparent within 48 hours, declining to approximately 30% by 2 weeks, accompanied by an increase in activation of the promitogenic transcription factor STAT3 (PY-STAT3). Immunofluorescence studies showed a selective loss of cav-1alpha and platelet endothelial cell adhesion molecule-1 in the PAEC layer within 48 hours after MCT but an increase in PY-STAT3. PAECs with cav-1alpha loss displayed high PY-STAT3 and nuclear immunostaining for proliferating cell nuclear antigen (PCNA). Biochemical studies showed a loss of cav-1alpha from the detergent-resistant lipid raft fraction concomitant with hyperactivation of STAT3. Moreover, cultured PAECs treated with MCT-pyrrole for 48 hours developed megalocytosis associated with hypo-oligomerization and reduction of cav-1alpha, hyperactivation of STAT3 and ERK1/2 signaling, and stimulation of DNA synthesis., Conclusions: MCT-induced disruption of cav-1alpha chaperone and scaffolding function in PAECs likely accounts for diverse alterations in endothelial cell signaling in this model of PH.

Interleukin-6 (IL-6) initiates STAT3 signaling in plasma membrane rafts with the subsequent transit of Tyr-phosphorylated STAT3 (PY-STAT3) through the cytoplasmic compartment to the nucleus in association with accessory proteins. We initially identified caveolin-1 (cav-1) as a candidate STAT3-associated accessory protein due to its co-localization with STAT3 and PY-STAT3 in flotation raft fractions, and heat shock protein 90 (HSP90) due to its inclusion in cytosolic STAT3-containing 200-400-kDa complexes. Subsequent immunomagnetic bead pullout assays showed that STAT3, PY-STAT3, cav-1, and HSP90 interacted in plasma membrane and cytoplasmic complexes derived from uninduced and stimulated Hep3B cells. This was a general property of STAT3 in that these interactions were also observed in alveolar epithelial type II-like cells, lung fibroblasts, and pulmonary arterial endothelial cells. Exposure of Hep3B cells to the raft disrupter methyl-beta-cyclodextrin for 1-10 min followed by IL-6 stimulation for 15 min preferentially inhibited the appearance of PY-STAT3 in the cav-1-enriched sedimentable cytoplasmic fraction, suggesting that these complexes may represent a trafficking intermediate immediately downstream from the raft. Because IL-6 is known to function in the body in the context of fever, the possibility that HSP90 may help preserve IL-6-induced STAT3 signaling at elevated temperature was investigated. Geldanamycin, an HSP90 inhibitor, markedly inhibited IL-6-stimulated STAT3 signaling in Hep3B hepatocytes cultured overnight at 39.5 degrees C as evaluated by DNA-shift assays, trafficking of PY-STAT3 to the nucleus, cross-precipitation of HSP90 by anti-STAT3 polyclonal antibody, and reporter/luciferase construct experiments. Taken together, the data show that IL-6/raft/STAT3 signaling is a chaperoned pathway that involves cav-1 and HSP90 as accessory proteins and suggest a mechanism for the preservation of this signaling during fever.

Left ventricular geometry is suspected to affect heterogeneity of myocardial repolarization; therefore, it is plausible but unproven that increased sphericity of the left ventricle is associated with greater QT interval dispersion. In 60 patients with dilated cardiomyopathy with left ventricular ejection fraction < or = 30%, we found that spherical distortion of the left ventricle was associated with increased QT dispersion, implying increased heterogeneity of myocardial repolarization.

Background: It is suspected that effective therapies are often underutilized in black compared with white patients with coronary artery disease (CAD)., Hypothesis: We hypothesized that an unfavorable bias may exist against black patients in the medical management of heart failure., Methods: In 566 consecutive adult subjects who were discharged alive from the hospital with a principal discharge diagnosis of heart failure, we assessed the effect of patient race on utilization of classes of medications (angiotensin-converting enzyme inhibitors [ACEI], digitalis, diuretic agents) and combinations of medications (effective vasodilators, i.e., ACEI or combined hydralazine and nitrate; effective combination therapy, i.e., effective vasodilator with digitalis and diuretic) known to be beneficial in symptomatic heart failure., Results: Compared with black patients (n = 182), white patients were older, had a higher incidence of coronary artery disease, lower incidence of hypertension, and lower serum creatinine and left ventricular end-diastolic diameter. In crude analyses, the utilization of all medications was similar between white and black patients. After adjustment for clinical differences, black patients were more likely to receive ACEI (adjusted odds ratio [OR] = 1.84; 95% confidence interval [CI] 1.13-3.01), effective vasodilators (OR = 1.97; CI 1.20-3.23), and effective combination therapy (OR = 1.66; CI 1.02-2.69) than white patients at the time of discharge from the hospital. No multivariate association was seen between patient race and use of digoxin or diuretics. In an analysis of subsets of patients with ejection fraction < 45% (n = 260), no association was seen between patient race and utilization of effective medical therapy., Conclusion: Our results show no unfavorable bias against black patients with decompensated heart failure.

Glucose-regulated protein 58 (GRP58/ER-60/ERp57), best known as a chaperone in the endoplasmic reticulum lumen, was previously identified by us as one of several accessory proteins in the S100 cytosol fraction of human hepatoma Hep3B cells that was differentially coshifted by anti-Stat3 antibody in an antibody-subtracted differential protein display assay. In the present study, the association between GRP58 and Stat3 in different cytoplasmic compartments was evaluated using cross-immunoprecipitation and cell-fractionation techniques. In the S100 cytosol fraction, three different anti-GRP58 polyclonal antibodies (pAb) cross-immunoprecipitated Stat3 (but not Stat1), and, conversely, anti-Stat3 pAb cross-immunoprecipitated GRP58. Both cytosolic Stat3 and GRP58 eluted during Superose-6 gel-filtration chromatography in complexes of size 200-400 kDa (statosome I), and anti-Stat3 pAb cross-immunoprecipitated GRp58 from these FPLC elution fractions. Using differential sedimentation and density equilibrium flotation methods, Stat3 and GRP58 were observed to be coassociated with cytoplasmic membranes enriched for the plasma membrane marker 5' nucleotidase but not with those containing the endoplasmic reticulum marker BiP/GRP78. The Stat3 and GRP58-containing plasma membrane fraction also contained Stat1, Stat5b, and gp130. Stat activation by orthovanadate caused the accumulation of PY-Stat3 in the GRP58-containing plasma membrane fraction. However, this PY-Stat3 was DNA-binding deficient. Likewise, excess exogenous recombinant human GRP58 prepared using a baculovirus expression system preferentially inhibited Stat3 DNA-binding activity in the S100 cytosol, suggesting that GRP58 may sequester activated Stat3. The new data confirm the association between GRP58 and Stat3 in cytosolic 200-400-kDa statosome I complexes and show that both GRP58 and Stat family members coassociate in the plasma membrane compartment. We suggest that the chaperone GRP58 may regulate signaling by sequestering inactive and activated Stat3.

STAT transcription factors signal from the plasma membrane to the nucleus in response to growth factors and cytokines. We have investigated whether plasma membrane "rafts" are involved in cytokine-activated STAT signaling. Cytokine-free human hepatoma Hep3B cells or cells treated with interleukin-6 (IL-6) or orthovanadate (a general activator of STATs) were fractionated, and plasma membrane raft fractions were obtained by equilibrium sedimentation or flotation through discontinuous sucrose gradients using either non-detergent or detergent-based (saponin or Triton X-100) methods. By Western blotting the plasma membrane raft fractions obtained using either non-detergent or detergent-based methods contained significant amounts of STAT1 and STAT3 (up to approximately 10% of the total cytoplasmic amount) as well as the integral raft proteins caveolin-1 and flotillin-1, the IL-6-receptor signal transducing chain gp130, the interferon-gamma receptor alpha chain (IFN-gammaRalpha), and the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57). Upon activation of signaling by IL-6 or orthovanadate the respective Tyr-phosphorylated STAT species were now also observed in the membrane raft fraction but in a form deficient in DNA binding. The data show pre-association of STATs with plasma membrane rafts in flotation fractions, which also contained caveolin-1 and flotillin-1, and suggest that Tyr phosphorylation may not in itself be sufficient to cause the departure of PY-STATs from plasma membrane rafts. Methyl-beta-cyclodextrin, which sequesters cholesterol and disrupts plasma membrane rafts, markedly inhibited IL-6- and IFN-gamma-induced STAT signaling. Signaling through specialized raft microdomains may be a general mechanism operating at the level of the plasma membrane through which cytokines and growth factors activate STAT species (the "raft-STAT signaling hypothesis").