New insights into the antioxidant activity of hydroxycinnamic and hydroxybenzoic systems: spectroscopic, electrochemistry, and cellular studies.

A series hydroxycinnamic and gallic acids and their derivatives were studied with the aim of evaluating their in vitro antioxidant properties both in homogeneous and in cellular systems. It was concluded from the oxygen radical absorbance capacity-fluorescein (ORAC-FL), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and cyclic voltammetry data that some compounds exhibit remarkable antioxidant properties. In general, in homogeneous media (DPPH assay), galloyl-based cinnamic and benzoic systems (compounds 7-11) were the most active, exhibiting the lowest oxidation potentials in both dimethyl sulfoxide (DMSO) and phosphate buffer. Yet, p-coumaric acid and its derivatives (compounds 1-3) disclosed the highest scavenging activity toward peroxyl radicals (ORAC-FL assay). Interesting structure-property- activity relationships between ORAC-FL, or DPPH radical, and redox potentials have been attained, showing that the latter parameter can be a valuable antioxidant measure. It was evidenced that redox potentials are related to the structural features of cinnamic and benzoic systems and that their activities are also dependent on the radical generated in the assay. Electron spin resonance data of the phenoxyl radicals generated both in DMSO and phosphate buffer support the assumption that radical stability is related to the type of phenolic system. Galloyl-based cinnamic and benzoic ester-type systems (compounds 9 and 11) were the most active and effective compounds in cell-based assays (51.13 ± 1.27% and 54.90 ± 3.65%, respectively). In cellular systems, hydroxycinnamic and hydroxybenzoic systems operate based on their intrinsic antioxidant outline and lipophilic properties, so the balance between these two properties is considered of the utmost importance to ensure their performance in the prevention or minimization of the effects due to free radical overproduction.

Type II diabetes mellitus and menopause: a multinational study.

BACKGROUND: Type II diabetes mellitus causes metabolic changes that may lead to early menopause and worsen climacteric symptoms. OBJECTIVES: To determine the risk factors for type II diabetes mellitus and assess the impact of this disease on the age of menopause and on climacteric symptoms. METHODS: A total of 6079 women aged between 40 and 59 years from 11 Latin American countries were requested to answer the Menopause Rating Scale and Goldberg Anxiety-Depression Scale. RESULTS: The prevalence of diabetes was 6.7%. Diabetes mellitus was associated with arterial hypertension (odds ratio (OR) 4.49; 95% confidence interval (CI) 3.47-5.31), the use of psychotropic drugs (OR 1.54; 95% CI 1.22-1.94), hormonal therapy (OR 1.46; 95% CI 1.11-1.92), ≥ 50 years of age (OR 1.48; 95% CI 1.17-1.86), overweight or obese (OR 1.47; 95% CI 1.15-1.89), and waist circumference ≥ 88 cm (OR 1.32; 95% CI 1.06-1.65). Factors associated with lower risk of diabetes were the use of hormonal contraceptives (OR 0.55; 95% CI 0.35-0.87), alcohol (OR 0.73; 95% CI 0.54-0.98) and living in cities > 2500 meters above sea level (OR 0.70; 95% CI 0.53-0.91) or with high temperatures (OR 0.67; 95% CI 0.51-0.88). In turn, diabetes tripled the risk of menopause in women under 45 years of age. Diabetes did not increase the risk of deterioration of quality of life due to climacteric symptoms. CONCLUSION: Menopause does not increase the risk of type II diabetes mellitus. Diabetes is associated with early menopause in women under 45 years of age.

Análisis de la prescripción inducida en una comarca de atención primaria / Analysis of the induced prescription in a primary care region

Objetivos. Conocer la prevalencia, el origen y el gasto atribuible de la prescripción inducida (PI) en atención primaria (AP) en la Comarca Oeste de Gipuzkoa, determinar el grado de acuerdo de los médicos de AP con respecto a la PI y analizar la adecuación de la PI a los indicadores del contrato de gestión clínica de la AP. Material y métodos. Diseño: estudio descriptivo, transversal y multicéntrico. Ámbito: AP, 38 médicos pertenecientes a 17 unidades de AP de la Comarca Oeste de Gipuzkoa. Participantes: prescripciones farmacéuticas financiables realizadas durante 2 días en consulta a demanda y crónicas generadas por el programa informático Osabide. Variables analizadas: tipo de prescripción, origen, especialidad del prescriptor, diagnóstico, precio y grado de acuerdo. Resultados. Se realizaron 6.919 prescripciones y el 44% fueron PI (intervalo de confianza del 95%: 42,8–45,1). El 62,2% del gasto total se atribuyó a la PI, con un precio medio por receta de 22,3€ para la PI y de 10,6€ para la prescripción propia. Los subgrupos terapéuticos de mayor gasto fueron los hipolipidemiantes y los broncodilatadores. Resultados. El grado de desacuerdo de los médicos participantes con la PI fue del 28,8%. La adecuación de los indicadores de calidad de la prescripción fue mayor en la prescripción propia que en la PI. Conclusiones. Existe un porcentaje elevado de PI asociado a un gasto elevado que se atribuye a la AP. El porcentaje de desacuerdo en AP con respecto a la PI es importante. Se observa una influencia elevada de la PI en la evaluación de los indicadores de calidad establecidos en la AP(AU)

Objectives. To find out the prevalence, origin and cost associated with Induced Prescription (IP) in Primary Health Care (PHC) in the West of Gipuzkoa (WG). To find out the extent to which PHC doctors agree with IP. To analyse the adaptation of IP to PHC clinical management contract indicators. Materials and methods. Design descriptive multi-centre cross-study. Location. Primary Health Care, 38 doctors from 17 WG PHC units. Participants. Pharmaceutical prescriptions eligible for finance over a period of two days in outpatients and chronic diseases generated by the Osabide computer application. Participants. Variables analysed: type of prescription, origin, prescriber, diagnosis, price and level of agreement. Results. A total of 6.919 prescriptions were made out, with 44% (95% CI: 42.8–45.1) being IP. Of the total cost, 62.2% was put down to IP, with an average price per prescription of €22.3,and in non-induced prescription (NIP) it was €10.62. The therapeutic subgroups with the highest cost were lipid lowering and bronchodilator drugs. The level of disagreement of the doctors taking part in IP was 28.8%. The adaptation to the quality indicators of the prescription was higher in NIP than in IP. Conclusions. There is a high percentage of IP associated with high costs attributed to PHC. The percentage of disagreement in PHC with regard to IP is significant. There is a high influence of IP on the evaluation of the quality indicators established in PHC(AU)

[Analysis of the induced prescription in a primary care region]. / Análisis de la prescripción inducida en una comarca de atención primaria.

OBJECTIVES: To find out the prevalence, origin and cost associated with Induced Prescription (IP) in Primary Health Care (PHC) in the West of Gipuzkoa (WG). To find out the extent to which PHC doctors agree with IP. To analyse the adaptation of IP to PHC clinical management contract indicators. MATERIALS AND METHODS: Design descriptive multi-centre cross-study. LOCATION: Primary Health Care, 38 doctors from 17 WG PHC units. PARTICIPANTS: Pharmaceutical prescriptions eligible for finance over a period of two days in outpatients and chronic diseases generated by the Osabide computer application. Variables analysed: type of prescription, origin, prescriber, diagnosis, price and level of agreement. RESULTS: A total of 6.919 prescriptions were made out, with 44% (95% CI: 42.8-45.1) being IP. Of the total cost, 62.2% was put down to IP, with an average price per prescription of €22.3,and in non-induced prescription (NIP) it was €10.62. The therapeutic subgroups with the highest cost were lipid lowering and bronchodilator drugs. The level of disagreement of the doctors taking part in IP was 28.8%. The adaptation to the quality indicators of the prescription was higher in NIP than in IP. CONCLUSIONS: There is a high percentage of IP associated with high costs attributed to PHC. The percentage of disagreement in PHC with regard to IP is significant. There is a high influence of IP on the evaluation of the quality indicators established in PHC.

Development of a methodology for the simultaneous determination of inorganic and organolead compounds using supercritical fluid extraction followed by gas chromatography-mass spectrometry and its application to environmental matrices.

A method for the extraction of triethyl lead (TEL(+)), trimethyl lead (TML(+)), and Pb(2+) from sand was developed using supercritical modified CO(2)-CH(3)OH extraction and in situ complexation with sodium diethyldithiocarbamate (NaDDTC) using a 2(5) factorial exploratory design is described. The screened variables were (i) pressure (69-193 bar), (ii) temperature (40-150 degrees C), (iii) ligand amount (0-100 mg), (iv) methanol volume (0.0-0.5 mL) and (v) static time (0-45 min). The optimum extraction conditions found were as follow: pressure, 193 bar; temperature, 40 degrees C; amount of NaDDTC, 100 mg; methanol volume, 0.5 mL; static time 45 min; and CO(2) flow rate, 1 mL min(-1). Under these conditions the following recoveries were obtained (TML(+) 97+/-2%, TEL(+) 70+/-5%, and Pb(2+) 100+/-4%). The presence of NaDDTC is not necessary for the extraction of TML(+) and TEL(+), but it is a very significative parameter for Pb(2+). A second experimental design 2(2)+star for temperature and pressure was realized, but the results were not better than those of the first model. SFE extract derivatization was achieved with pentylmagnesium bromide, and target analyte determination was carried out by gas chromatography-mass spectrometry. Detection limits in the full-scan mode were 4, 10, and 39 pg as lead for TMPeL, TEPeL and PbPe(4), respectively. The method was validated with urban dust containing TML(+) (CRM 605. Pb 7.9 +/-1.2 microg kg(-1)) and river sediment containing inorganic lead (GBW08301. Pb 79.0+/-12.0 mg kg(-1)) as reference materials. The proposed method was applied to lead analysis in sand collected from an oil-polluted beach in Chile.

Phosphorylation of class I MHC molecules in the absence of phorbol esters is an intracellular event and may be characteristic of trafficking molecules.

Class I Major Histocompatibility Complex (MHC) molecules are displayed at the cell surface where they present antigenic peptides to T lymphocytes. Class I MHC molecules undergo cytoplasmic domain phosphorylation on a serine residue late in their biosynthesis. Here we show that phosphorylation occurs on mature, beta(2)-microglobulin-associated class I MHC molecules in a mouse lymphoid cell line. Both recently synthesized class I MHC molecules and molecules which are at least 3 h old become phosphorylated. Approximately 14% of phosphorylated class I MHC molecules occur at the cell surface. Density gradient analysis indicates that phosphorylated class I MHC molecules also occur in lamp(+) intracellular compartments and in fractions containing rab4, a GTP-binding protein associated with recycling endosomes. Class I MHC molecules are endocytosed and recycled to the cell surface in these cells. Furthermore, the lysosomotropic drug, primaquine, inhibits both class I MHC phosphorylation and its recycling back to the cell surface, suggesting that phosphorylation is related to class I MHC recycling. These observations are intriguing since several studies have shown that class I MHC molecules can acquire antigenic peptides in NH(4)Cl-sensitive compartments. Hence, class I MHC phosphorylation may play a role in regulating intracellular sorting through these compartments.

Selective export of MHC class I molecules from the ER after their dissociation from TAP.

It has been assumed that upon dissociation from TAP, MHC class I molecules exit the ER by nonselective bulk flow. We now show that exit must occur by association with cargo receptors. Inconsistent with exit by bulk flow, loading of MHC class I molecules with high-affinity peptides triggers dissociation from TAP but has no effect on rates of ER-to-Golgi transport. Moreover, peptide-loaded MHC class I molecules accumulate at ER exit sites from which TAP molecules are excluded. Consistent with receptor-mediated exit, ER-to-Golgi transport of MHC class I molecules is independent of their cytoplasmic tails, which themselves lack ER export motifs. In addition, we show that MHC class I molecules associate with the putative cargo receptor BAP31.

Endosomal/lysosomal retention and degradation of major histocompatibility complex class I molecules is induced by myxoma virus.

The highly immunosuppressive leporipoxvirus myxoma, previously was shown to promote the loss of cell surface class I major histocompatibility complex (MHC I) molecules. Here, we show that myxoma virus induces the loss of both cell surface and intracellular post-Golgi, beta(2)-microglobulin-associated MHC I. Myxoma-induced loss of these MHC I molecules is abrogated by vacuolar ATPase inhibitors, NH(4)Cl, and leupeptin. Furthermore, immunofluorescence microscopic studies reveal that in myxoma-infected cells, beta(2)-microglobulin-associated MHC I accumulates in Lamp-1(+) vesicular structures, suggesting that myxoma virus targets MHC I for degradation in late endosomes and/or lysosomes. These events are regulated by early gene product or products because they occur unabated in cells infected with myxoma virus in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. Studies with baby green monkey kidney cells transfected with wild-type and tail-less forms of a mouse MHC I molecule, H-2L(d), indicate that the MHC I cytoplasmic tail is required for myxoma-induced localization in Lamp-1(+) organelles. Myxoma-induced endocytosis and degradation of MHC I may provide the virus with a means of dispensing with cell surface MHC I molecules that were loaded with peptides derived from viral proteins synthesized early in infection.

Ab initio association with beta 2-microglobulin during biosynthesis of the H-2Ld class I major histocompatibility complex heavy chain promotes proper disulfide bond formation and stable peptide binding.

In vitro translation studies indicate that the beta 2-microglobulin (beta 2-m) light chain influences the formation of intrachain disulfide bonds in class I major histocompatibility complex (MHC) molecules during their biosynthesis. We now have examined the influence of beta 2-m on class I MHC intrachain disulfide bond formation in vivo. Using beta 2-m+ and beta 2-m- derivatives of a cell line transfected with the mouse H-2Ld gene, we show that all of the H-2Ld molecules from beta 2-m+ cells have both the alpha 2 and alpha 3 intrachain disulfide bonds, whereas about 50% of the H-2Ld molecules from beta 2-m- cells have only one of these bonds. All of the free H-2Ld heavy chains from beta 2-m+ cells can undergo a peptide-induced conformational change and can bind exogenous peptide and beta 2-m stably in vitro. Only those H-2Ld molecules from beta 2-m- cells, which have both intrachain disulfide bonds, undergo a peptide- and beta 2-m-induced conformational change in vitro. These H-2Ld molecules do not bind beta 2-m and peptide stably in vitro. From these results emerges a greater understanding of the role of beta 2-m at the time of class I MHC molecule synthesis: beta 2-m promotes intrachain disulfide bond formation in the class I MHC molecule and additionally affects class I MHC structure to render it competent to form stable trimolecular complexes with peptide and beta 2-m.

Class I histocompatibility molecule association with phosphorylated calnexin. Implications for rates of intracellular transport.

Recent studies have shown that the endoplasmic reticulum (ER)-resident protein, calnexin, associates with class I major histocompatibility complex (MHC) molecules early in their biosynthesis. It has been suggested that calnexin participates in the assembly of class I MHC molecules or in the retention within the ER of unassembled class I molecules. We have examined the role of phosphorylation of calnexin in its association with mouse class I MHC molecules. We show that phosphocalnexin associates with H-2Ld and H-2Db molecules but not with H-2Kb and H-2Dd molecules, although calnexin-H-2Kb association can be demonstrated. These observations are interesting in light of the fact that H-2Kb and H-2Dd molecules are transported out of the ER more rapidly than are H-2Ld and H-2Db molecules. H-2Ld and H-2Db molecules differ in amino acid sequence only in their membrane-distal alpha 1 and alpha 2 domains. Nevertheless, the affinity of phosphocalnexin for H-2Ld is greater than its affinity for H-2Db. Furthermore, H-2Db becomes endoglycosidase H-resistant more slowly in cells in which it associates with phosphocalnexin than in cells in which it does not. Ca2+ ionophore A23187 prevents association of phosphocalnexin with H-2Ld molecules in vivo but does not cause the disruption of phosphocalnexin-H-2Ld complexes after they have formed. A23187 does not prevent assembly of H-2Ld-beta 2-microglobulin (beta 2-m) heterodimers. Furthermore, phosphocalnexin is found associated with H-2Ld molecules regardless of their state of assembly with beta 2-m and antigenic peptide. These results suggest that phosphocalnexin association with class I MHC molecules does not play a role in assembly of the class I MHC-beta 2-m-peptide complex nor in preventing release of unassembled class I molecules from the ER but may otherwise influence their rate of transport through the ER.

Truncation mutants define and locate cytoplasmic barriers to lateral mobility of membrane glycoproteins.

The lateral mobility of cell membrane glycoproteins is often restricted by dynamic barriers. These barriers have been detected by measurements of fluorescence photobleaching and recovery (FPR) and barrier-free path (BFP). To define the location and properties of the barriers, we compared the lateral mobility, measured by FPR and BFP, of wild-type class I major histocompatibility complex (MHC) membrane glycoproteins with the lateral mobility of mutant class I MHC glycoproteins truncated in their cytoplasmic domains. Mutants with 0 or 4 residues in the cytoplasmic domain were as mobile as lipid-anchored class I MHC molecules, molecules whose lateral mobility is relatively unrestricted by barriers. In contrast, mobility of class I MHC molecules with 7-residue cytoplasmic domains was as restricted as mobility of class I molecules with full-length, 31-residue cytoplasmic domains. Though some of the difference between the mobilities of mutants with 4- or 0-residue domains and the other class I molecules may be due to differences in the net charge of the cytoplasmic domain, FPR measurements of the mobility of molecules with 7-residue domains show that length of the cytoplasmic domain has an important influence on the lateral mobility. Model calculations suggest that the barriers to lateral mobility are 2-3 nm below the membrane bilayer.

The cytoplasmic domain of the H-2Ld class I major histocompatibility complex molecule is differentially accessible to immunological and biochemical probes during transport to the cell surface.

An antiserum was generated against a synthetic peptide corresponding to a portion of the cytoplasmic domains of the H-2Ld and H-2Db class I major histocompatibility complex molecules of the mouse. This antibody preparation, R4, binds exclusively to endoglycosidase H-resistant H-2Ld/Db molecules which are not associated with beta 2-microglobulin. Interestingly, acquisition of resistance to endoglycosidase H precedes acquisition of R4 reactivity by 30 min. R4-reactive H-2Ld and H-2Db molecules occur on the cell surface and are phosphorylated in vivo. Other studies show that the tyrosine in the cytoplasmic domain is accessible to radioiodination on only a subset of H-2Ld molecules, and that the two-dimensional electrophoretic profiles of phosphorylated H-2L/Db molecules, of R4-reactive molecules, and of H-2Ld molecules radiolabeled on this cytoplasmic domain tyrosine are virtually identical. R4-reactive H-2Ld molecules do not undergo the peptide- and beta 2-microglobulin-induced conformational changes characteristic of free class I major histocompatibility complex heavy chains. The accessibility of the H-2Ld cytoplasmic domain to R4 and to radioiodination late in biosynthesis and its biological significance are discussed.

[Perception of patients from rural areas on referral resolution]. / Percepción del paciente de áreas rurales sobre la resolución de las interconsultas.

Eighty two patients from two public rural outpatient clinics were interviewed about referrals requested during June 1991, the unresolved proportion after six months, the alternative ways used to solve the health problems and their perception about this issue. A total of 95 referrals were requested in June in these clinics (4 every 100 consultations) and information about 85 was obtained. The frequency varies from 10% in the women's program, most of the referrals are directed to surgery, respiratory diseases and dermatology, in the adult's program, to radiology, gastroenterology and a wide variety of specialties. Six months later almost all the referrals had an appointment at the reference hospital. In spite of this, 20 patients did not receive the information and five sought a solution with private physicians. Of the 65 remaining patients, 12.3% lost their appointment due to diverse reasons such as preferring private physicians, oblivion or lack of money. Fifty seven patients went to the reference hospital. Thirty eight percent of these felt that their health problem had not been solved at the moment of the study, because they were still in treatment, were waiting a bed in surgery, the results of some laboratory tests were delayed or they perceived bad treatment. It is concluded that only half of the patients referred to specialists, felt that their health problem had been solved six months later.

In vivo dimeric association of class I MHC heavy chains. Possible relationship to class I MHC heavy chain-beta 2-microglobulin dissociation.

Class I MHC molecules have been thought to occur in vivo both as class I MHC heavy chain-beta 2-m heterodimers, which are or are not associated with antigenic peptide, and as free class I MHC heavy chains. Class I MHC molecules are now found also to occur in another type of structure: a heavy chain-heavy chain dimer. Biochemical studies show that heavy chain dimers are disulfide-linked via a conserved cytoplasmic domain cysteine. H-2Ld, H-2Db, and H-2Dd class I dimers fail to react with certain alpha 1 and alpha 2 domain-specific antibodies. Furthermore, although beta 2-m-specific antibodies coprecipitate class I MHC heavy chains, they do not coprecipitate class I MHC heavy chain dimers. Pulse-chase studies show that heavy chain dimer formation occurs at different points in the biosynthesis of class I MHC molecules in beta 2-m+ and beta 2-m- cells: in beta 2-m+ cells, heavy chain dimers form after the class I molecules have traversed the medial Golgi cisternae, whereas in beta 2-m- cells they form immediately. Culturing of beta 2-m+ cells with exogenous beta 2-m prevents the formation of H-2Ld/Db heavy chain dimers. We conclude that dimer formation occurs as a consequence of loss or unavailability of beta 2-m. Class I MHC heavy chain dimerization may provide a mechanism for removal of immunologically dysfunctional molecules.

[Adult morbidity consultations in rural medical centers]. / Morbilidad consultada por los adultos en postas y estaciones medico rurales.

A sample of 823 adult morbidity consultations in four rural medical centers in the VI Region in Chile were studied according to age and group of diagnosis. There was a reduction in the number of consultations with age. The following were the most frequent diagnoses: respiratory disorders 17.3%, infections and parasitic infestation 10.2%, circulatory diseases 10.6%, gastrointestinal diseases 9.8%, genitourinary diseases 8.9%, musculoskeletal disorders 8.6% and mental illnesses 8.0%. No differences were found between this pattern of morbidity and that found in similar studies in urban areas. Important differences were found between the rural centres included in the study and these should be analysed in more detail, as they may reveal different risk factors for these populations.

A double-labeling method for measuring induction of protein phosphorylation.

Protein phosphorylation is widely believed to play a regulatory role in signal transduction, mitosis, cell proliferation, cell motility, cell shape, gene regulation, and many other cellular processes. Thus, the quantitation of phosphorylation of specific cellular proteins may provide insight into the mechanisms by which phosphorylation is employed in regulation. Moreover, identification of phosphorylation substrates of various cellular kinases provides an important first step in determining their role in cellular regulation. However, accurate measurement of the differential phosphorylation of cellular proteins under different physiological conditions is often difficult to achieve. To address this problem, we have developed an in vivo double-labeling protocol (utilizing [3H]-, [14C]-, or [35S]-radiolabeled amino acids and [32P]-orthophosphate) that allows the quantitation of the amount of specific phosphorylation of a given protein from densitometric analysis of autoradiograms of polyacrylamide gels. This double-labeling strategy provides a means of quantitating the phosphorylation of individual biosynthetically labeled proteins. This method can be used in the analysis of immunoprecipitated proteins, proteins from subcellular fractions, such as nuclei or selected membrane fractions, or even total cellular proteins displayed on two-dimensional gels.

Endocytosis of the class I major histocompatibility antigen via a phorbol myristate acetate-inducible pathway is a cell-specific phenomenon and requires the cytoplasmic domain.

Class I major histocompatibility (MHC) antigens are expressed by virtually all mammalian cells, yet their levels of expression and behavior on the cell surface vary in a cell-specific fashion. A panel of lymphoid (both B and T) and nonlymphoid cell lines was used to study the kinetics of internalization of the H-2Ld class I MHC in different cell types. These studies revealed that endocytosis of H-2Ld occurs by both constitutive and PMA-regulated pathways in lymphoid cells, but only by a PMA-refractory pathway in the nonlymphoid cells tested. Transfectant derivatives of the T lymphoma, EL4, which express wild-type or mutant H-2Ld class I MHC antigens, were used to investigate the requirement for the cytoplasmic domain of the class I MHC antigen for its endocytosis in T lymphocytes. These studies showed that modification or deletion of the cytoplasmic domain of H-2Ld abrogates endocytosis via a PMA-regulated pathway. The role of cytoplasmic domain phosphorylation in PMA-inducible endocytosis was examined. The wild-type H-2Ld antigen is phosphorylated in all cell types examined, and this phosphorylation is up-regulated by PMA treatment. In contrast, cytoplasmic domain mutants of H-2Ld fail to be phosphorylated in vivo, in the presence or absence of PMA. The universality of PMA-inducible hyperphosphorylation of the class I MHC antigen among diverse cell types leads us to conclude that phosphorylation of the cytoplasmic domain, while perhaps necessary, is not sufficient for triggering endocytosis via a PMA-inducible pathway. Furthermore, the results with the cytoplasmic domain mutants of H-2Ld suggest that a structural conformation of the class I MHC cytoplasmic domain is required for endocytosis via this route.

Clonal variation in cell surface display of an H-2 protein lacking a cytoplasmic tail.

Truncated variants of the gene encoding H-2Ld, an integral membrane protein encoded by the major histocompatibility complex, were constructed by in vitro mutagenesis to elucidate the function of charged amino acids found on the cytoplasmic side of the transmembrane (TM) region. Analysis of cloned L cells transfected with these genes shows that the seven amino acids following the TM segment, four of which are basic, enhance the cell surface expression of H-2Ld protein but are not required for it. However, some clones do not express a tailless H-2Ld protein on the cell surface but express it intracellularly where it has a long half-life. Turnover measurements on cell surface H-2Ld proteins suggest that the basic residues following the TM segment are not a "stop transfer" sequence (Blobel, G., 1980, Proc. Natl. Acad. Sci. USA., 77:1496-1500) which anchors the H-2Ld protein in the membrane. Pulse-chase and endoglycosidase H sensitivity studies show that H-2Ld proteins lacking some or all of the basic residues and H-2Ld proteins which have a full-length cytoplasmic tail are processed with different kinetics. These results suggest an involvement of the membrane-proximal region of the cytoplasmic tail in the intracellular transport of H-2Ld. We further suggest that the L cell clones which do and do not express a tailless H-2Ld protein on the cell surface differ in the ability to transport a tailless integral membrane protein to the cell surface.