Melting during late-stage rifting in Afar is hot and deep.

Investigations of a variety of continental rifts and margins worldwide have revealed that a considerable volume of melt can intrude into the crust during continental breakup, modifying its composition and thermal structure. However, it is unclear whether the cause of voluminous melt production at volcanic rifts is primarily increased mantle temperature or plate thinning. Also disputed is the extent to which plate stretching or thinning is uniform or varies with depth with the entire continental lithospheric mantle potentially being removed before plate rupture. Here we show that the extensive magmatism during rifting along the southern Red Sea rift in Afar, a unique region of sub-aerial transition from continental to oceanic rifting, is driven by deep melting of hotter-than-normal asthenosphere. Petrogenetic modelling shows that melts are predominantly generated at depths greater than 80 kilometres, implying the existence of a thick upper thermo-mechanical boundary layer in a rift system approaching the point of plate rupture. Numerical modelling of rift development shows that when breakup occurs at the slow extension rates observed in Afar, the survival of a thick plate is an inevitable consequence of conductive cooling of the lithosphere, even when the underlying asthenosphere is hot. Sustained magmatic activity during rifting in Afar thus requires persistently high mantle temperatures, which would allow melting at high pressure beneath the thick plate. If extensive plate thinning does occur during breakup it must do so abruptly at a late stage, immediately before the formation of the new ocean basin.

Early-life gut microbiome and egg allergy.

BACKGROUND: Gut microbiota may play a role in egg allergy. We sought to examine the association between early-life gut microbiota and egg allergy. METHODS: We studied 141 children with egg allergy and controls from the multicenter Consortium of Food Allergy Research study. At enrollment (age 3 to 16 months), fecal samples were collected, and clinical evaluation, egg-specific IgE measurement, and egg skin prick test were performed. Gut microbiome was profiled by 16S rRNA sequencing. Analyses for the primary outcome of egg allergy at enrollment, and the secondary outcomes of egg sensitization at enrollment and resolution of egg allergy by age 8 years, were performed using Quantitative Insights into Microbial Ecology, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, and Statistical Analysis of Metagenomic Profiles. RESULTS: Compared to controls, increased alpha diversity and distinct taxa (PERMANOVA P = 5.0 × 10-4 ) characterized the early-life gut microbiome of children with egg allergy. Genera from the Lachnospiraceae, Streptococcaceae, and Leuconostocaceae families were differentially abundant in children with egg allergy. Predicted metagenome functional analyses showed differential purine metabolism by the gut microbiota of egg-allergic subjects (Kruskal-Wallis Padj  = 0.021). Greater gut microbiome diversity and genera from Lachnospiraceae and Ruminococcaceae were associated with egg sensitization (PERMANOVA P = 5.0 × 10-4 ). Among those with egg allergy, there was no association between early-life gut microbiota and egg allergy resolution by age 8 years. CONCLUSION: The distinct early-life gut microbiota in egg-allergic and egg-sensitized children identified by our study may point to targets for preventive or therapeutic intervention.

Long-term follow-up of fenestrated endovascular repair for juxtarenal aortic aneurysm.

BACKGROUND: Fenestrated endovascular aneurysm repair (FEVAR) is increasingly being used for juxtarenal aortic aneurysms. The aim of this study was to review long-term results and assess the importance of changing stent-graft design on outcomes. METHODS: This was a retrospective review of all patients who underwent FEVAR within a single unit over 12 years (February 2003 to December 2015). Kaplan-Meier analysis of survival, and freedom from target vessel loss, aneurysm expansion, graft-related endoleak and secondary intervention was performed. Comparison between outcomes of less complex grafts (fewer than 3 fenestrations) and more complex grafts (3 or 4 fenestrations) was undertaken. RESULTS: Some 173 patients underwent FEVAR; median age was 76 (i.q.r. 70-79) years and 90·2 per cent were men. Median aneurysm diameter was 63 (59-71) mm and median follow-up was 34 (16-50) months. The adjusted primary technical operative success rate was 95·4 per cent. The in-hospital mortality rate was 5·2 per cent; there was no known aneurysm-related death during follow-up. Median survival was 7·1 (95 per cent c.i. 5·2 to 8·1) years and overall survival was 60·1 per cent (104 of 173). There was a trend towards an increasing number of fenestrations in the graft design over time. In-hospital mortality appeared higher when more complex stent-grafts were used (8 versus 2 per cent for stent-grafts with 3-4 versus fewer than 3 fenestrations; P = 0·059). Graft-related endoleaks were more common following deployment of stent-grafts with three or four fenestrations (12 of 90 versus 6 of 83; P < 0·001). CONCLUSION: Fenestrated endovascular aneurysm repair for juxtarenal aneurysm is associated with few aneurysm-related deaths in the long term. Significant numbers of secondary interventions are required, but the majority of these can be performed using an endovascular approach.

Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs.

Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog.

Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs.

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.

Component-resolved analysis of IgA, IgE, and IgG4 during egg OIT identifies markers associated with sustained unresponsiveness.

BACKGROUND: In a previously reported CoFAR study, 55 subjects with egg allergy underwent randomized, placebo-controlled egg oral immunotherapy (eOIT). Active treatment induced desensitization in most and sustained unresponsiveness (SU) in a smaller subset. We hypothesized that component-resolved analysis of IgE, IgG4, IgA, IgA1, and IgA2 may identify potential biomarkers of SU in OIT subjects. METHODS: Longitudinal samples for 51 egg-allergic subjects (37 active and 14 placebo) were available. Egg white (EW)-, ovalbumin (OVA)-, and ovomucoid (OVM)-specific levels of IgA, IgA1, and IgA2 were quantified by ELISA. IgE and IgG4 to these antigens were quantified using ImmunoCAP® . Clinical responders achieved SU to egg; all others were considered nonresponders. Between-group comparisons were made among active and placebo, as well as responders and nonresponders. RESULTS: No placebo subjects achieved responder status. Through month 48, among the 37 active subjects, baseline IgE-OVM was lower in responders (median 3.97 kU/l, n = 19) than in nonresponders (10.9 kU/l, n = 18, P = 0.010). Logistic regression analysis revealed that lower baseline IgE-EW (P = 0.038), IgE-OVM (P = 0.032), and a higher IgG4/IgE-OVM ratio (P = 0.013) were associated with clinical response. Relative increases in IgG4-EW, IgA-EW, and IgA2-EW were observed in responders (P = 0.024, 0.024, and 0.029, respectively). IgG4/IgE, IgA/IgE, and IgA2/IgE ratios for EW and IgA/IgE ratio for OVA were found to be significantly elevated among responders (P = 0.004, 0.009, 0.028, and 0.008, respectively). CONCLUSIONS: Increased IgG4-EW, IgA-EW, and IgA2-EW during eOIT are associated with clinical response to eOIT. Lower pretreatment IgE-EW and IgE-OVM are also associated with SU. Future studies are needed to evaluate and validate these potential biomarkers.

Differential targeting of feline photoreceptors by recombinant adeno-associated viral vectors: implications for preclinical gene therapy trials.

The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.

Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach.

Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset, and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface-exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected; four dogs received intravitreal injections and eight dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal) and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach.

Chronic disease influences over 40 major life-changing decisions (MLCDs): a qualitative study in dermatology and general medicine.

BACKGROUND: Chronic disease not only impairs patients' psycho-social well-being but also influences major life-changing decisions (MLCDs). There is little information about the types of MLCDs affected and the long-term consequences. OBJECTIVES: The aims were to identify the MLCDs influenced by chronic disease, to define 'MLCD' and to suggest support strategies for patients taking MLCDs. METHODS: Adult dermatology patients explained how their chronic disease had influenced MLCDs in individual interviews. Adult patients from other medical specialities gave similar information by postal survey. NVivo8 software was used for qualitative analysis of data. Themes were categorized through a coding-recoding iterative process. RESULTS: There were 308 evaluable responses (male 55.2%; mean age = 51.8 years, mean disease duration = 19 years) from the 365 (55.7%) responses to the 655 patient invitations. These were used to generate themes to conceptualize 'MLCDs'. The most frequently reported MLCDs in the dermatology interviews concerned career choice (66%), job (58%), choice of clothing (54%), relationships (52%), education (44%), stopping swimming (34%), moving abroad (32%), not socializing (34%), wearing make-up (22%) and having children (22%). In the postal survey early retirement (40.6%), impact on job (29.4%), having children (24.8%), career choice (22.4%) and relationships (15.5%) were most commonly reported. The number of MLCDs reported by individuals was inversely related to age. Forty-one affected MLCD themes were grouped into 18 MLCD categories. A definition of MLCD was developed and strategies suggested to support patients. CONCLUSIONS: Chronic diseases influence a wide range of MLCDs. MLCDs are a novel domain in disease burden assessment. Clinicians' knowledge about this is important in patient management.

RPE65 gene therapy slows cone loss in Rpe65-deficient dogs.

Recent clinical trials of retinal pigment epithelium gene (RPE65) supplementation therapy in Leber congenital amaurosis type 2 patients have demonstrated improvements in rod and cone function, but it may be some years before the effects of therapy on photoreceptor survival become apparent. The Rpe65-deficient dog is a very useful pre-clinical model in which to test efficacy of therapies, because the dog has a retina with a high degree of similarity to that of humans. In this study, we evaluated the effect of RPE65 gene therapy on photoreceptor survival in order to predict the potential benefit and limitations of therapy in patients. We examined the retinas of Rpe65-deficient dogs after RPE65 gene therapy to evaluate the preservation of rods and cone photoreceptor subtypes. We found that gene therapy preserves both rods and cones. While the moderate loss of rods in the Rpe65-deficient dog retina is slowed by gene therapy, S-cones are lost extensively and gene therapy can prevent that loss, although only within the treated area. Although LM-cones are not lost extensively, cone opsin mislocalization indicates that they are stressed, and this can be partially reversed by gene therapy. Our results suggest that gene therapy may be able to slow cone degeneration in patients if intervention is sufficiently early and also that it is probably important to treat the macula in order to preserve central function.

Evidence of pathway-specific basophil anergy induced by peanut oral immunotherapy in peanut-allergic children.

BACKGROUND: In Westernized countries, over 1% of the population is allergic to peanuts or tree nuts, which carries a risk of severe allergic reactions. Several studies support the efficacy of peanut oral immunotherapy (OIT) for reducing the clinical sensitivity of affected individuals; however, the mechanisms of this effect are still being characterized. One mechanism that may contribute is the suppression of effector cells, such as basophils. Basophil anergy has been characterized in vitro as a pathway-specific hyporesponsiveness; however, this has not been demonstrated to occur in vivo. OBJECTIVE: To evaluate the hypothesis that basophil anergy occurs in vivo due to chronic allergen exposure in the setting of a clinical oral immunotherapy trial. METHODS: Samples of peripheral blood were obtained from subjects during a placebo-controlled clinical trial of peanut OIT. Basophil reactivity to in vitro stimulation with peanut allergen and controls was assessed by the upregulation of activation markers, CD63 and CD203c, measured by flow cytometry. RESULTS: The upregulation of CD63 following stimulation of the IgE receptor, either specifically with peanut allergen or non-specifically with anti-IgE antibody, was strongly suppressed by active OIT. However, OIT did not significantly suppress this response in basophils stimulated by the distinct fMLP receptor pathway. In the subset of subjects with egg sensitization, active peanut OIT also suppressed CD63 upregulation in response to stimulation with egg allergen. Allergen OIT also suppressed the upregulation of CD203c including in response to stimulation with IL-3 alone. CONCLUSION: Peanut OIT induces a hyporesponsive state in basophils that is consistent with pathway-specific anergy previously described in vitro. This suggests the hypothesis that effector cell anergy could contribute to clinical desensitization.

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function.

The purpose of this study was to evaluate whether immune responses interfered with gene therapy rescue using subretinally delivered recombinant adeno-associated viral vector serotype 2 carrying the RPE65 cDNA gene driven by the human RPE65 promoter (rAAV2.hRPE65p.hRPE65) in the second eye of RPE65-/- dogs that had previously been treated in a similar manner in the other eye. Bilateral subretinal injection was performed in nine dogs with the second eye treated 85-180 days after the first. Electroretinography (ERG) and vision testing showed rescue in 16 of 18 treated eyes, with no significant difference between first and second treated eyes. A serum neutralizing antibody (NAb) response to rAAV2 was detected in all treated animals, but this did not prevent or reduce the effectiveness of rescue in the second treated eye. We conclude that successful rescue using subretinal rAAV2.hRPE65p.hRPE65 gene therapy in the second eye is not precluded by prior gene therapy in the contralateral eye of the RPE65-/- dog. This finding has important implications for the treatment of human LCA type II patients.

Growth-factor-dependent mitogenesis requires two distinct phases of signalling.

Prolonged and continuous exposure to growth factors is required to commit cells to the cell cycle. Here we show that the prolonged requirement for growth factor can be replaced with two short pulses of mitogen. The first pulse of growth factor moves the cell through the initial segment of the G0 to S interval. This initial pulse also makes cells responsive to a second pulse of growth factor, which engages components of the cell-cycle machinery necessary for progression into S phase. We also show that activation of MAP kinase kinase (MEK) and induction of the transcription factor c-Myc are sufficient to drive the first, but not the second, phase of signalling. Furthermore, synthetic phosphatidylinositol-3-OH kinase (PI(3)K) lipid products are sufficient to drive the second phase of signalling, but not the first. These findings suggest that there is a common signalling cascade by which mitogens drive arrested cells into the cell cycle, and that this cascade involves the temporally coordinated input of MEK, c-Myc and PI(3)K.

The Ebola virus ribonucleoprotein complex: a novel VP30-L interaction identified.

The ribonucleoprotein (RNP) complex of Ebola virus (EBOV) is known to be a multiprotein/RNA structure, however, knowledge is rather limited regarding the actual protein-protein interactions involved in its formation. Here we show that singularly expressed VP35 and VP30 are present throughout the cytoplasm, while NP forms prominent cytoplasmic inclusions and L forms smaller perinuclear inclusions. We could demonstrate the existence of NP-VP35, NP-VP30 and VP35-L interactions, similar to those described for Marburg virus (MARV) based on the redistribution of protein partners into NP and L inclusion bodies. Significantly, a novel VP30-L interaction was also identified and found to form as part of an NP-VP30-L bridge structure, similar to that formed by VP35. The identification of these interactions allows a preliminary model of the EBOV RNP complex structure to be proposed, and may provide insight into filovirus transcriptional regulation.

AAV retinal transduction in a large animal model species: comparison of a self-complementary AAV2/5 with a single-stranded AAV2/5 vector.

PURPOSE: To compare self-complementary (sc) and single-stranded (ss) adeno-associated viral 2/5 (AAV2/5) vectors for retinal cell transduction in the dog when delivered by subretinal injection. METHODS: ScAAV2/5 and ssAAV2/5 vectors encoding enhanced green fluorescent protein (GFP) under control of the chicken beta actin promoter were prepared to the same titer. Equal amounts of viral particles were delivered into the subretinal spaces of both eyes of two dogs. In each dog, one eye received the scAAV2/5 and the other the ssAAV2/5. In vivo expression of GFP was monitored ophthalmoscopically. The dogs were sacrificed, and their retinas were examined by fluorescent microscopy and immunohistochemistry to determine GFP expression patterns and to assay for glial reactivity. RESULTS: GFP expression in the scAAV2/5 injected eyes was detectable at a much earlier time point than in the ssAAV2/5 injected eyes. Expression of GFP was also at higher levels in the scAAV2/5-injected eyes. Expression levels remained stable for the seven month duration of the study. The types of cells transduced by both vectors were similar; there was strong reporter gene expression in the RPE and photoreceptors, although not all cones in the transduced area expressed GFP. Some horizontal and Müller cells were also transduced. CONCLUSIONS: When delivered by subretinal injection in the dog, scAAV2/5 induces faster and stronger transgene expression than ssAAV2/5. The spectrum of retinal neurons transduced is similar between the two vectors. These results confirm in a large animal model those previously reported in the mouse. ScAAV2/5 shows promise for use in the treatment of conditions where a rapid transgene expression is desirable. Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.

Phasing rectangular apertures.

Several techniques have been developed to phase apertures in the context of astronomical telescopes with segmented mirrors. Phasing multiple apertures, however, is important in a wide range of optical applications. The application of primary interest in this paper is the phasing of multiple short pulse laser beams for fast ignition fusion experiments. In this paper analytic expressions are derived for parameters such as the far-field distribution, a line-integrated form of the far-field distribution that could be fit to measured data, enclosed energy or energy-in-a-bucket and center-of-mass that can then be used to phase two rectangular apertures. Experimental data is taken with a MEMS device to simulate the two apertures and comparisons are made between the analytic parameters and those derived from the measurements. Two methods, fitting the measured far-field distribution to the theoretical distribution and measuring the ensquared energy in the far-field, produced overall phase variance between the 100 measurements of less than 0.005 rad(2) or an RMS displacement of less than 12 nm.

Phosphatidylinositol 3-kinase is required for the formation of constitutive transport vesicles from the TGN.

An 85-kD cytosolic complex (p62(cplx)), consisting of a 62-kD phosphoprotein (p62) and a 25-kD GTPase, has been shown to be essential for the cell-free reconstitution of polymeric IgA receptor (pIgA-R)-containing exocytic transport vesicle formation from the TGN (Jones, S.M., J.R. Crosby, J. Salamero, and K.E. Howell. 1993. J. Cell Biol. 122:775-788). Here the p62(cplx) is identified as a regulatory subunit of a novel phosphatidylinositol 3-kinase (PI3-kinase). This p62(cplx)-associated PI3-kinase activity is stimulated by activation of the p62(cplx)-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62(cplx)-associated PI3-kinase activity in TGN-derived vesicle formation is indicated by the finding that both lipid kinase activity and the formation of pIgA-R-containing exocytic vesicles from the TGN are inhibited by wortmannin with similar dose-response curves and 50% inhibitory concentrations (3.5 microM). These findings indicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62(cplx)-associated PI3-kinase and an activated GTPase are the essential molecules that drive production of this PI(3)P.

A cytosolic complex of p62 and rab6 associates with TGN38/41 and is involved in budding of exocytic vesicles from the trans-Golgi network.

TGN38/41, an integral membrane protein predominantly localized to the trans-Golgi network, has been shown to cycle to the plasma membrane and return to the TGN within 30 min. (Ladinsky, M. S., and K. E. Howell. 1992. Eur. J. Cell Biol. 59:92-105). In characterizing the proteins which associate with TGN38/41, a peripheral 62-kD protein, two forms of rab6 and two other small GTP-binding proteins were identified by coimmunoprecipitation. However, approximately 90% of the 62-kD protein is cytosolic and is associated with the same subset of small GTP-binding proteins. Both the membrane and cytoplasmic complexes were characterized by sizing column fractionation and velocity sedimentation. The membrane complex was approximately 250 kD (11.6 S) consisting of the cytosolic complex and a heterodimer of TGN38/41 (160 kD). The cytosolic complex was approximately 86 kD (6.1 S) consisting of p62 and one small GTP-binding protein. Preliminary evidence indicates that phosphorylation of the p62 molecule regulates the dissociation of the cytosolic complex from TGN38/41. Functionally the cytosolic p62 complex must bind to TGN38/41 for the budding of exocytic transport vesicles from the TGN as assayed in a cell-free system (Salamero, J., E. S. Sztul, and K. E. Howell. 1990. Proc. Natl. Acad. Sci. USA. 87:7717-7721). Interference with p62, rab6 or TGN38, and TGN41 cytoplasmic domains by immunodepletion or competing peptides completely inhibited the budding of exocytic transport vesicles. These results support an essential role for interaction of the cytosolic p62/rab6 complex with TGN38/41 in budding of exocytic vesicles from the TGN.