The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions.

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.

Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells.

Cells from the bone marrow can present peptides that are derived from tumors, transplants, and self-tissues. Here we describe how dendritic cells (DCs) process phagocytosed cell fragments onto major histocompatibility complex (MHC) class II products with unusual efficacy. This was monitored with the Y-Ae monoclonal antibody that is specific for complexes of I-Ab MHC class II presenting a peptide derived from I-Ealpha. When immature DCs from I-Ab mice were cultured for 5-20 h with activated I-E+ B blasts, either necrotic or apoptotic, the DCs produced the epitope recognized by the Y-Ae monoclonal antibody and stimulated T cells reactive with the same MHC-peptide complex. Antigen transfer was also observed with human cells, where human histocompatibility leukocyte antigen (HLA)-DRalpha includes the same peptide sequence as mouse I-Ealpha. Antigen transfer was preceded by uptake of B cell fragments into MHC class II-rich compartments. Quantitation of the amount of I-E protein in the B cell fragments revealed that phagocytosed I-E was 1-10 thousand times more efficient in generating MHC-peptide complexes than preprocessed I-E peptide. When we injected different I-E- bearing cells into C57BL/6 mice to look for a similar phenomenon in vivo, we found that short-lived migrating DCs could be processed by most of the recipient DCs in the lymph node. The consequence of antigen transfer from migratory DCs to lymph node DCs is not yet known, but we suggest that in the steady state, i.e., in the absence of stimuli for DC maturation, this transfer leads to peripheral tolerance of the T cell repertoire to self.

Cloning, expression, and localization of a novel gamma-adaptin-like molecule.

We describe the cloning, expression, and localization of gamma2-adaptin, a novel isoform of gamma-adaptin. The predicted human and mouse gamma2-adaptin proteins are approximately 90 kDa and 64.4% and 61.7%) identical to gamma-adaptin, respectively. gamma2-Adaptin was localized to the Golgi, its localization distinct from gamma-adaptin. The membrane association of gamma- and gamma2-adaptin could further be distinguished by differential sensitivity to the fungal metabolite brefeldin A, gamma2-adaptin binding being insensitive to drug treatment. Together, these results suggest that gamma2-adaptin plays a role in membrane transport distinct from that played by gamma-adaptin.

Sensitivity and positive predictive value of Medicare Part B physician claims for rheumatologic diagnoses and procedures.

OBJECTIVE: To examine the sensitivity and positive predictive value of Medicare physician claims for select rheumatic conditions managed in rheumatology specialty practices. METHODS: Eight rheumatologists in 3 states abstracted 378 patient office records to obtain information on diagnosis and office procedures. The Medicare Part B physician claims for these patient visits were obtained from the Health Care Financing Administration. The sensitivity of the claims data for a specific diagnosis was calculated as the proportion of all patients whose office records for a particular visit documented that diagnosis and who also had physician claims for that visit which identified that diagnosis. The positive predictive value was evaluated in a separate sample of 331 patient visits identified in Medicare physician claims. The positive predictive value of the claims data for a specific diagnosis was calculated as the proportion of patients with that diagnosis coded in the claims for a particular visit who also had the diagnosis documented in the medical record for that visit. RESULTS: Ninety percent of abstracted office medical records were matched successfully with Medicare physician claims. The sensitivity of the Medicare physician claims was 0.90 (95% confidence interval [CI] 0.85-0.95) for rheumatoid arthritis (RA), 0.85 (95% CI 0.73-0.97) for systemic lupus erythematosus (SLE), and 0.85 (95% CI 0.78-1.0) for aspiration or injection procedures. The sensitivity for osteoarthritis (OA) of the hip or knee was < or = 0.50 if 5-digit codes specifying anatomic site were required. The sensitivity for fibromyalgia (FM) was 0.48 (95% CI 0.28-0.68). The positive predictive values were at least 0.90 for RA, SLE, and aspiration or injection procedures. Positive predictive values for FM and the 5-digit site-specific codes for OA of the knee were 0.83 (95% CI 0.66-1.0) and 0.88 (95% CI 0.75-1.0), respectively, while the positive predictive value of the 5-digit site-specific codes for OA of the hip was zero (95% CI 0-0.26). The positive predictive value of OA at any site was 0.83 (95% CI 0.76-0.90). CONCLUSION: In specialty practice, Medicare physician claims had high sensitivity and positive predictive value for RA, SLE, OA without specification of anatomic site, and injection or aspiration procedures. The claims had lower sensitivity and predictive value for FM and for OA of the hip. The accuracy of Medicare physician claims for other conditions and in the primary care setting requires further investigation.

Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component epsilon-COP.

Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit epsilon-COP. In addition to exhibiting conditional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes redistributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable epsilon-COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant phenotypes were also corrected by transfection of wild-type epsilon-COP cDNA demonstrating that they directly or indirectly reflected the epsilon-COP defect. Taken together, the results suggest that epsilon-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.

Biochemical heterogeneity and phosphorylation of coatomer subunits.

The coat protomer complex I (COPI) family of coat proteins are involved in the assembly of membrane-associated coats thought to mediate vesicular transport between the endoplasmic reticulum and the Golgi complex, between adjacent Golgi cisternae, and possibly in the endocytic pathway. We investigated whether this heterogeneity in the sites of COPI action might be reflected in biochemical heterogeneity of one or more COPI subunits. A simplified method was devised to purify the cytosolic COPI precursor complex, coatomer, from rat liver cytosol. The individual subunits were analyzed by high resolution two dimensional gel electrophoresis and mass spectroscopic analysis of tryptic peptides. Considerable charge heterogeneity was observed, particularly for the beta-COP and delta-COP subunits. The multiple species detected, however, did not appear to reflect the presence of distinct translation products but rather a significant degree of protein phosphorylation. The observed pI of beta-COP was sensitive to alkaline phosphatase digestion. Moreover, isolation of coatomer from metabolically labeled tissue culture cells demonstrated directly that both beta-COP and delta-COP, but no other coatomer subunits, were serine-phosphorylated. COPI phosphorylation may regulate coatomer assembly, membrane recruitment, or the specificity of coatomer-organelle interaction.

Cytoplasmic coat proteins involved in endosome function.

Endosomes are intermediates for a complex series of sorting and transport events that occur during receptor-mediated endocytosis. These involve the recognition of targeting determinants on the cytoplasmic domains of many membrane proteins as well as the formations of specific transport vesicles. Accordingly, endosome function is likely to be governed by the regulated assembly of cytoplasmic coat complexes. We have found that, in vitro, endosomes recruit a characteristic set of cytoplasmic proteins in a GTP gamma S-stimulated and brefeldin A-sensitive fashion. Among these are members of the COP-I and ARF families of coat proteins. In addition, endosomes were also found to assemble distinct, clathrin-like coats. Since microinjection of antibodies to beta-COP inhibits the entry of enveloped viruses via the endocytic pathway, it is apparent that the recruitment of COP-I or COP-I-related proteins plays an important role in the function of endosomes in intact cells.

Isolation and characterization of the rat liver actin N-acetylaminopeptidase.

Actins from most eukaryotes undergo a unique post-translational modification of the amino terminus called "processing." Processing consists of the removal of an amino-terminal Ac-Met or Ac-Cys to leave an acidic amino-terminal residue. We have previously demonstrated that this reaction is not catalyzed by the ribosomally associated methionine aminopeptidase or by other previously described acetylaminopeptidases. Here we present the isolation and characterization of the actin N-acetylaminopeptidase (ANAP) from rat liver. A five-step purification protocol achieves a 4100-fold purification of the enzyme with an overall 8% recovery of activity. ANAP is a 77-kDa monomer with a pI of 4.6. Using unprocessed yeast actin as a substrate, the Km of ANAP is 3.5 microM. Purified ANAP was used to generate a polyclonal antibody. The antibody has been used along with activity assays to demonstrate the presence of ANAP in a variety of rat tissues. Finally, evidence is presented that in mammals, ANAP may function with a second, as yet unpurified, component to process actin amino termini.

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.

Unusual metabolism of the yeast actin amino terminus.

In this paper we have examined the post-translational modifications of the NH2 terminus of actin from the yeast Saccharomyces cerevisiae. Like actins examined previously, this actin contains an acetylated NH2 terminus. Actins in other organisms undergo a unique post-translational processing event in which the initial amino acid(s) are removed by an actin-specific processing enzyme in an acetylation-dependent reaction. This is defined as actin processing. In yeast, actin retains its initiator Met in vivo and is thus not processed even though a rat liver actin processing enzyme can process yeast actin in vitro. This lack of actin processing appears to be a general property of fungi, as the actin from three other species, Aspergillus nidulans, Schizosaccharomyces pombe, and Candida albicans are not NH2 terminally processed either. Yeast actin is a class I actin; its initiator Met directly precedes an acidic residue. We converted yeast actin to a class II species by inserting a Cys codon between the Met-1 and Asp-2 codons. In normal class II actins the Cys residue is removed as acetyl-Cys during processing. Neither the mutant actin nor chick beta-actin (a class I actin) are processed when expressed in yeast. S. cerevisiae thus appears to be also incapable of processing exogenous actins. Further study of the mutant actin containing a Cys at position 2 shows that 30-40% of this actin is stably unacetylated. This unacetylated actin does not have a shorter half-life than the acetylated form. From these studies we conclude that 1) NH2-terminal actin-specific processing is not required for actin function in yeast and three other fungi, 2) yeast are apparently incapable of processing any type of actin precursor, and 3) the stability of a yeast pseudo-class II actin is not affected by the acetylation state of the NH2 terminus.

Hypodense eosinophils and interleukin 5 activity in the blood of patients with the eosinophilia-myalgia syndrome.

The recent recognition of the eosinophilia-myalgia syndrome (EMS) associated with the ingestion of L-tryptophan prompted an analysis of the peripheral blood eosinophil phenotypes and of the serum eosinophil hematopoietins in this disorder. Five patients with an illness characterized by the abrupt onset of aching skeletal muscles, edema, thickening and induration of the skin, and marked blood eosinophilia associated with L-tryptophan ingestion provided eosinophils, serum, or both, for evaluation. Gradient sedimentation density analysis of the peripheral blood eosinophils from four of these patients revealed that 43 +/- 13% (mean +/- SEM) of the cells had converted to the abnormal (hypodense) sedimenting phenotype. When normodense eosinophils from the reference donors were cultured for 3 days in medium supplemented with increasing concentrations of serum from the patients with EMS, their viability increased in a dose-dependent manner to 45%, which was significantly augmented over the effect of normal serum. This eosinophil viability-sustaining activity was inhibited by 76 +/- 7% (mean +/- SEM; n = 3) by the addition of anti-interleukin 5 (IL-5) but not by neutralizing antibodies monospecific for either granulocyte/macrophage colony-stimulating factor (GM-CSF) or IL-3. IL-5, an eosinophilopoietic factor, converts normodense peripheral blood eosinophils in vitro to a hypodense sedimenting form with extended viability and augmented biologic responses to activating stimuli. Thus, the presence of IL-5 in the sera of patients with EMS may contribute to the development and maintenance of the eosinophilia and may regulate the conversion of the peripheral blood eosinophils to the hypodense phenotype with augmented pathobiologic potential.

Identification of N-acetylmethionine as the product released during the NH2-terminal processing of a pseudo-class I actin.

Genes for the various isoactins define two classes of actin. Class I actin genes code for Met-Asp(Glu)-actin, and class II actin genes code for Met-X-Asp(Glu)-actin where X is usually cysteine. Amino termini of both are removed in an acetylation-dependent processing reaction yielding acetyl-Asp(Glu)-actin. Both classes are processed at approximately equal rate (t1/2 = 15 min) in vivo. In vitro, class II actins are 90% processed by endogenous enzymes after 60 min in a rabbit reticulocyte lysate system, whereas class I actins are only minimally processed during this period. Using site-directed mutagenesis of a human skeletal muscle isoactin coupled with in vitro transcription and translation methods, we have synthesized a pseudo-class I actin in which the penultimate cysteine has been changed to an aspartic acid, thus placing a class I amino terminus on an otherwise class II actin molecule. The pseudo-class I actin was less than 20% processed during the translation period as determined by peptide mapping. It was further processed by exogenous processing enzyme at a rate compatible with a class I actin. These results indicate that the major actin determinant controlling differential actin-processing rates is the amino-terminal residue being cleaved, not the remaining structure of the actin molecule. We have also demonstrated for the first time that N-acetylmethionine is the immediately released product from the amino terminus of a pseudo-class I actin during processing.

Disseminated aspergillosis and pacemaker endocarditis.

Clinical and pathologic findings in a 65-year old woman with fever of unknown origin are described in this report. Generalized aspergillosis with endocarditis was demonstrated at autopsy. The patient had no recognized risk factors for the development of fungal infection. A functional transvenous pacemaker lead, inserted 2 years previously, was completely encased in a large infected thrombus and may have been the initial site of infection. Septicemia and endocarditis are rare but well-described complications of cardiac pacing, and should be considered in the differential diagnosis of fever of unknown origin in patients with pacemakers.