Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains.

GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.

Evidence supporting a late Golgi location for lactosylceramide to ganglioside GM3 conversion.

Ganglioside GM2 synthase and other enzymes required for complex ganglioside synthesis were localized recently to the trans Golgi network (TGN). However, there are conflicting reports as to the location of GM3 synthase; originally this enzyme was detected in the early Golgi of rat liver but a recent report localized it to the late Golgi. We have used chimeric forms of ganglioside GM2 synthase to determine if the location of lactosylceramide (LacCer) to GM3 conversion in Chinese hamster ovary (CHO) cells was the early or late Golgi. Our approach tested whether GM3 could be utilized as a substrate by GM2 synthase chimeras which were targeted to compartments earlier than the trans Golgi, i.e., GM3 produced in the cis Golgi should be utilized by GM2 synthase located anywhere in the Golgi whereas GM3 produced in the trans Golgi should only be used by GM2 synthase located in the trans Golgi or TGN. Comparison of cell lines stably expressing these chimeras revealed that the in vivo functional activity of GM2 synthase decreased progressively as the enzyme was targeted to earlier compartments; specifically, the percentage of GM3 converted to GM2 was 83-86% for wild type enzyme, 70% for the medial Golgi targeted enzyme, 13% for the ER and cis Golgi targeted enzyme, and only 1.7% for the ER targeted enzyme. Thus, these data are consistent with a late Golgi location for LacCer to GM3 conversion in these cells.

Reevaluating the effect of Brefeldin A (BFA) on ganglioside synthesis: the location of GM2 synthase cannot be deduced from the inhibition of GM2 synthesis by BFA.

Brefeldin A reversibly disassembles the Golgi complex, causing mixing of the Golgi cisternae with the ER while the trans Golgi network persists as part of a separate endosomal membrane system. Because of this compartmental separation, Brefeldin A treatment has been used to map the sub-Golgi locations of several Golgi enzymes including GM2 synthase. We previously proposed that GM2 synthase might be located in a distal portion of the Golgi complex which in the presence of Brefeldin A would be separated from the substrate ganglioside GM3 present in the mixed ER-Golgi membrane system. In the present study we show using GM2 synthase chimeras that GM2 synthesis was blocked by Brefeldin A when GM2 synthase was distributed throughout all Golgi subcompartments or even when it was restricted to the medial Golgi. Because these findings opposed our speculation regarding a distal location of this enzyme, we sought an alternative explanation for the inhibition of ganglioside synthesis by Brefeldin A. However, Brefeldin A did not degrade GM2 synthase, prevent its homodimerization, or inhibit its in vitro activity. Brefeldin A did result in the conversion of a portion of membrane bound GM2 synthase into a soluble form which has minimal capability to produce GM2 in whole cells. However, this conversion was not sufficient to explain the nearly total loss of GM2 production in intact cells in the presence of Brefeldin A. Nevertheless, the results of this study indicate that Brefeldin A-induced inhibition of ganglioside synthesis cannot be used to deduce the location of GM2 synthase.

Relationship of provider characteristics to outcomes, process, and costs of care for community-acquired pneumonia.

OBJECTIVES: The authors describe the relation of provider characteristics to processes, costs, and outcomes of medical care for elderly patients hospitalized for community-acquired pneumonia. METHODS: Using Medicare claims data, Medicare beneficiaries discharged from Pennsylvania hospitals during 1990 with community-acquired pneumonia were identified. Claims data were used to ascertain mortality, readmissions, use of procedures and physician consultations, and the costs of care. The relationship of these measures to provider characteristics was analyzed using regression techniques to adjust for patient characteristics, including comorbidity and microbial etiology. RESULTS: Among 22,294 pneumonia episodes studied, 30-day mortality was 17.0%. After adjusting for patient characteristics, 30-day mortality and readmission rates were unrelated to hospital teaching status or urban location or to physician specialty. Use of procedures and physician consultations was more common and costs were 11% higher among patients discharged from teaching hospitals compared with nonteaching hospitals. Similarly, costs were 15% higher at urban hospitals compared with rural hospitals. General internists and medical subspecialists used more procedures and had higher costs than family practitioners. CONCLUSIONS: Processes and costs of care for community-acquired pneumonia varied by provider characteristics, but neither mortality nor readmission rates did. These differences cannot be explained by clinical variables in the database. Further studies should determine whether less costly patterns of care for pneumonia, and perhaps other conditions, could replace more costly ones without compromising patient outcomes.

Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts.

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3-galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.

Wide-area network connecting a hospital drug informatics center with a university.

A wide-area network (WAN) connecting a new drug informatics center in a university-affiliated hospital with the university's campus-based computer network is described. In 1994 a pharmacy school developed a drug informatics center in an affiliated hospital. The center was originally designed around a local-area network (LAN) to be located at the hospital and planned to provide clients with easy access to typical productivity software and various electronic information resources. Only occasional modem connections to the university network were envisioned. However, large price increases in information retrieval systems and decreases in the cost of a frame relay connection (T1 line) to the campus network led to the installation of a WAN when the drug informatics center was established. Technical, political, and legal problems were overcome, and the connection was made. The WAN gave faculty and students at the hospital access to many of the university's computing and Internet resources. In addition, the faculty and students have access to various files and programs available only on the drug informatics center's file server at the affiliated hospital. It cost about $6500 to install all WAN equipment and maintain the frame relay for the first year, or a third of what would have been necessary for information retrieval software had a separate LAN been established at the hospital. A WAN connecting a drug informatics center and a university's computer network gave the center access to more electronic information resources at lower cost than would have been possible with a separate LAN.

Beta 1,4 N-acetylgalactosaminyltransferase (GM2/GD2/GA2 synthase) forms homodimers in the endoplasmic reticulum: a strategy to test for dimerization of Golgi membrane proteins.

Many Golgi membrane-bound glycosyltransferases exist as intermolecular disulfide bonded species, some of which have been demonstrated to be homodimers. Evidence for homodimer formation has come primarily from radiation inactivation experiments. We utilized an alternative strategy to test for homodimer formation of the cloned beta 1,4 N-acetylgalactosaminyltransferase (GalNAcT) responsible for synthesis of the glycosphingolipids GM2, GD2, and GA2. We stably transfected CHO cells with myc epitopetagged GalNAcT, which localizes primarily to the Golgi, and a hemagglutinin (HA) epitope-tagged GalNAcT fusion protein in which the cytoplasmic domain of GalNAcT was replaced by an ER retention signal. We then sought evidence for dimer formation between the two forms of GalNAcT. Immunoprecipitation with anti-myc or anti-HA co-immunoprecipitated the HA-tagged form or the myc-tagged form, respectively, providing evidence for the physical association of the two forms of GalNAcT. As a result of this association, GalNAcT/myc increased in the ER as demonstrated by Western blots and immunofluorescence. The rapid formation of dimers provided further evidence for dimer formation occurring in the ER. In summary, these results demonstrate that GalNAcT forms homodimers as a result of intermolecular disulfide bond formation in the ER. Furthermore, this ER motif strategy is potentially useful for demonstrating homodimer formation of other Golgi enzymes.

Community-acquired pneumonia: can it be defined with claims data?

The use of administrative data to study pneumonia is limited because International Classification of Diseases, 9th revision, Clinical Modification (ICD9-CM) diagnosis codes do not specify whether pneumonia is community-acquired (CAP), a key clinical distinction. We classified 212 patients discharged with a diagnosis code for pneumonia as to whether or not they had CAP, using three administrative data-based systems (Diagnosis Related Groups (DRGs) alone, principal diagnosis alone, and a complex algorithm). We examined agreement with classification by clinician chart review. We also compared the length of stay (LOS) and mortality among the CAP populations identified with different methods. Agreement between the clinical review and the three administrative data methods ranged from 86 to 80%. Classification by DRG performed least well. Populations defined by claims data had similar mortality but shorter mean LOS (9.70, 9.40, and 7.91 days for the algorithm, principal diagnosis and DRG methods, respectively) than the clinically defined population (10.85 days). We conclude that studies of CAP using populations identified by claims may underestimate LOS.

Appearance of beta 1,4 N-acetylgalactosaminyltransferase (glycosphingolipids GA2/GM2/GD2 synthase) in embryonic chicken vitreous humor during development.

PURPOSE: beta 1,4 N-Acetylgalactosaminyltransferase (GalNAcT) is a type II integral membrane protein of the Golgi apparatus that catalyzes the synthesis of the glycosphingolipids GM2, GD2, and GA2. The activity of GalNAcT in chick retinal cells increases 6-fold between embryonic days 7 and 14. Because GalNAcT, like many Golgi glycosyltransferases, is proteolytically cleaved from Golgi membranes to release a soluble form into the culture medium of cells transfected with the cloned human enzyme, we tested whether GalNAcT might be released from embryonic retinal cells into the vitreous humor. METHODS: Samples of vitreous humor and plasma and extracts of retinal cells were assayed for GalNAcT activity. RESULTS: The activity of a soluble form of GalNAcT in embryonic chick vitreous humor was nearly undetectable until embryonic day 10, then increased more than six fold until day 16, and remained at that level until birth. The activity was identified as authentic GalNAcT based on a requirement for Mn++, GSL substrate specificity, and product characterization. GalNAcT activity in embryonic plasma was roughly 10% that of the corresponding vitreous humor, suggesting that the plasma was not the source of the activity in the vitreous. CONCLUSIONS: GalNAcT in embryonic chicken vitreous humor is likely due either to a specific release from neural retinal cells or due to non-specific lysis of these cells during apoptosis associated with the development of the retina. Regardless of the source, GalNAcT in the vitreous humor has the potential to function as a lectin by binding to gangliosides GD3 and GM3 on the surface of retinal cells and, thereby, to influence neuronal development.

Beta1,4-N-acetylgalactosaminyltransferase (GM2 synthase) is released from Golgi membranes as a neuraminidase-sensitive, disulfide-bonded dimer by a cathepsin D-like protease.

Many Golgi membrane-bound glycosyltransferases are released from cells in a soluble form. To characterize this release process, we stably transfected Chinese hamster ovary cells with three myc epitope-tagged forms of cloned beta1, 4-N-acetylgalactosaminyltransferase (GalNAcT); two of these forms resided in the Golgi, while the third was retained in the ER. GalNAcT was released into the culture medium from cells transfected with the Golgi forms but not with the ER form of the enzyme. The medium from cells transfected with the Golgi forms contained disulfide-bonded dimers of GalNAcT, which carried neuraminidase sensitive, complex N-linked carbohydrate chains. This soluble species represented the major degradation product of cellular GalNAcT, which turned over with a half-time of about 1.7 h. The soluble species consisted of a mixture of truncated GalNAcT molecules, the major form of which was produced by cleavage near the boundary between the transmembrane and lumenal domains between Leu-23 and Tyr-24. This cleavage site fits the sequence pattern for sites cleaved by cathepsin D (van Noort, J.M., and van der Drift, A. C.M. (1989) J. Biol. Chem. 264, 14159-14164). These findings suggest that GalNAcT is converted from a membrane-bound to a soluble form as a result of cleavage by a cathepsin D-like protease in a compartment late in the Golgi secretory pathway.

Dissemination of clinical results. Mastectomy versus lumpectomy and radiation therapy.

OBJECTIVES: This study is an assessment of the extent to which clinical findings concerning mastectomy versus lumpectomy with radiation treatment have been disseminated in practice over time. METHODS: The authors examined the use of breast-conserving surgery followed by radiation therapy as an alternative treatment to mastectomy for early-stage breast cancer by analyzing 5 years (1986-1990) of inpatient and outpatient claims data from four insurers: Medicare, Medicaid, Blue Cross of Western Pennsylvania, and Pennsylvania Blue Shield. The 9,288 women who were eligible for either a lumpectomy or mastectomy during the study period represented approximately 90% of south western Pennsylvania's adult female population. Given the efficacy of both procedures, the authors expected a trend toward more BCS. RESULTS: By 1990, the use of lumpectomy increased significantly to 42.4% from 35.2%. The choice of lumpectomy was associated with younger women, private health insurance, absence of axillary node metastases, and treatment in urban hospitals. The authors also found, however, that only 45.3% of women with Medicaid coverage who had a lumpectomy during the study period received the requisite follow-up radiation therapy, compared with 77.5% of private insurance subscribers and 88.1% of Medicare beneficiaries. This finding is troubling even though there was substantially more compliance in the later years of the study, with 60.0% of eligible Medicaid beneficiaries receiving follow-up radiation therapy in 1990. CONCLUSIONS: This research illustrates the usefulness of administrative claims data in describing trends and practice patterns as well as the need for a different type of research to discover the reasons for the lack of compliance with treatment protocols by women or physicians.

Cloned beta 1,4N-acetylgalactosaminyltransferase: subcellular localization and formation of disulfide bonded species.

Cloned human beta 1,4N-acetylgalactosaminyltransferase (GalNAcT) catalyzes the synthesis of the glycosphingolipids GM2, GD2, and gangliotriosylceramide. To determine the subcellular location of this enzyme and whether it exists in intermolecular disulfide bonded species, we stably transfected Chinese hamster ovary (CHO) cells with three myc epitope-tagged forms of the GalNAcT gene: the native enzyme; the lumenal domain of GalNAcT fused to the cytoplasmic and transmembrane domains of N-acetylglucosaminyltransferase I (GNT); and the transmembrane and lumenal domains of GalNAcT fused to the cytoplasmic domain of the Iip33 form of human invariant chain in order to retain the enzyme in the endoplasmic reticulum (ER). Immunoelectron microscopic analysis with anti-myc revealed that GalNAcT/myc was present throughout the Golgi stack, the GNT/GalNAcT/myc form was restricted primarily to the medial Golgi cisternae, and the Iip33/GalNAcT/myc form was restricted to the ER. Cells transfected with each of the three constructs contained high levels of GM2 synthase activity in vitro, but only the GalNAcT/myc form and the GNT/GalNAcT/myc forms were able to synthesize the GM2 product in vivo. The enzyme produced by all three constructs was present in the transfected cells in a disulfide bonded form having a molecular size consistent with that of a homodimer or higher aggregate.

Are small firms greater health risks?

To test whether use of health care services is a function of firm size, we analyzed a three-year database (1988-1990) of private insurance claims, representing 28,990 firms and approximately 1.4 million subscribers in western Pennsylvania. In this database both small and large firms had higher medically underwritten costs than mid-size firms had. Furthermore, risk-pooling alternatives that included small companies had a lower cost per subscriber than the risk pools that included large companies, especially companies of more than 500 contract holders. Age, sex, health status, and the types of hospitals used for inpatient care of pooled subscribers, in combination, were found to be the important determinants of costs. With risk adjustment based on these factors to correct for adverse risk selection, community rating can be a feasible approach to increasing the affordability and accessibility of health insurance to the majority of those who lack it.

Cloned beta 1,4 N-acetylgalactosaminyltransferase synthesizes GA2 as well as gangliosides GM2 and GD2. GM3 synthesis has priority over GA2 synthesis for utilization of lactosylceramide substrate in vivo.

Earlier studies reached conflicting conclusions as to the ability of the beta 1,4 N-acetylgalactosaminyltransferase (GalNAc-T) that synthesizes gangliosides GM2 and GD2 to also produce gangliotriosylceramide (GA2). We constructed an experimental system in which to address this question. Wild type Chinese hamster ovary (CHO) cells contain ganglioside GM3 as the most complex glycosphingolipid (GSL), whereas the CHO glycosylation mutant Lec2, which is deficient in sialylation, accumulates lactosylceramide with little GM3 being produced. We transfected both cell types with a plasmid containing a cloned GalNAc-T. Whereas transfected CHO cells produced GM2 as the major complex GSL, the major product in transfected Lec2 cells was GA2. Both types of transfected cells but not the untransfected cells expressed the transfected gene and contained high levels of enzyme activity for synthesizing both GM2 and GA2 in vitro. In summary, these results indicate that this enzyme can in fact synthesize GA2 as well as GM2 and GD2. In addition, these findings suggest that in CHO cells the synthesis of GM3 in vivo has priority over GA2 synthesis for utilization of the substrate lactosylceramide, resulting in little GA2 being produced even though GalNAc-T is present and active. Thus, competition for substrate between glycosylation pathways may have profound effects on the GSL pattern of cells.

PMC Patient Severity Scale: derivation and validation.

OBJECTIVE: This study describes the derivation and validation of the Patient Management Category (PMC) Severity Scale, which provides a method of assessing the overall severity of a hospitalized patient's illnesses, based on the patient's unique clinical conditions, their interaction, and the resultant, combined risk of morbidity and mortality. DATA SOURCES: Derivation of the PMC Severity Scale was based on clinical judgment together with empirical analysis of more than a half million patients discharged from acute care hospitals in Maryland during 1989. The scale was validated by using two distinct calendar years (1988 and 1990) of patients data from the same Maryland hospitals and a six-month patient database from California (1990). STUDY DESIGN: The PMC Severity Scale is an ordinal scale with seven levels: Level 7 represents the greatest likelihood of death and major disease burden. The scale quantifies the severity of each of the patient's disease(s) and accounts for the effect of all coexisting conditions and complications. DATA EXTRACTION METHODS: Publicly available, statewide all-payer claims databases were acquired from Maryland and California. PRINCIPAL FINDINGS: The independent relationships between the PMC Severity Scale with mortality and with length of stay are statistically different across severity levels within each population tested, but the relationships are statistically similar over time. Further, the PMC Severity Scale was determined to be a stable predictor of mortality and LOS across two diverse geographic regions. CONCLUSIONS: Since the severity of a patient's illness is one of the factors that influences the outcomes of care, the PMC Severity Scale can be used successfully as a risk adjustment tool in a variety of quality applications.

Using Medicare claims for outcomes research.

Medicare claims databases have several advantages for use in constructing episodes of care for outcomes research. They are population-based, relatively inexpensive to obtain, include large numbers of cases, and can be used for long-term follow-up. However, the sheer size of these claims databases, along with their primarily administrative (as opposed to clinical) nature, requires that researchers take special care in using them. The 10 PORTs using Medicare claims provided information on their approach to several key issues in working with these data, including: 1) identifying the index cases or patient cohorts to be studied; 2) defining the length of the episode; and 3) measuring outcomes. This paper reports the experience and knowledge gained by these PORTs in using these claims to create and analyze episodes of care.