Migration and health service delivery.

Migration has positive and integrative effects on health service delivery. This paper presents initiatives promoting circular migration of diaspora health professionals to contribute to health service delivery and capacity development in their countries of origin. The paper will also highlight the contributions that foreign trained and foreign born health professionals can make to the delivery of migrant friendly health services for diverse multi-cultural populations.

Formation of RuvABC-Holliday junction complexes in vitro.

In Escherichia coli, the RuvA, RuvB and RuvC proteins are required for the late stages of homologous recombination and DNA repair. RuvA and RuvB form a complex that interacts with Holliday junctions--crossed DNA structures that are recombination intermediates--and promotes branch migration; RuvC is a junction-specific endonuclease that resolves Holliday junctions and completes the recombination process. Because genetic and biochemical experiments suggest that the processes of branch migration and resolution are linked, coimmunoprecipitation experiments were carried out to determine whether the three Ruv proteins interact to form a functional complex (RuvABC). Using a synthetic Holliday junction, a multisubunit complex containing the junction and RuvA, RuvB and RuvC was detected. In the absence of RuvB, RuvAC-junction complexes were observed. Complex formation was not facilitated by duplex DNA. The identification of a RuvABC-junction complex provides direct evidence that the RuvABC proteins interact at the Holliday junction.

Ethnic differences in the association between body mass index and impedance index (Ht2/Z) in adult women and men using a leg-to-leg bioimpedance method.

BACKGROUND: Ethnic differences in the association between body mass index (BMI) and body fat suggest that body composition varies across ethnic groups. OBJECTIVE: To investigate the association between impedance index - a measure of tissue resistivity - and BMI in adults of different ethnic groups (Asian Indians, West Africans and White Caucasians) living in their native countries. METHODS: Male (n=329) and female (n=277) adult subjects (18-50 years) living in urban areas in the UK, The Gambia and Pakistan were studied. Body weight and height were measured and BMI calculated. The same leg-to-leg bioimpedance instrument was used in each study and impedance index (height(2) (cm)/impedance (Omega)) used as measure of tissue resistivity. RESULTS: In women, Asian Indians and West Africans had a significantly greater increase in impedance index per unit increase in BMI compared with white Caucasians (P<0.001). In men, Asian Indians had a significantly lower impedance index compared with West Africans and white Caucasians (P<0.001). CONCLUSION: Different ethnic groups may have different tissue resistivity for the same BMI indicative of systematic differences in body composition.

A growth-dependent post-translational modification of annexin VI.

Annexin VI (p68, 67-kDa calelectrin) is a member of a family of Ca2+/phospholipid-binding proteins, that includes p35 (annexin I) and p36 (annexin II), the major cellular substrates for phosphorylation by the epidermal growth factor receptor and pp60v-src tyrosine kinase activities, respectively. We report here that like annexins I and II, annexin VI is phosphorylated in vivo, but that in contrast, annexin VI phosphorylation is associated with cell growth. In both Swiss 3T3 fibroblasts and human T-lymphoblasts the pattern of phosphorylation followed an almost identical profile. In particular, annexin VI was not phosphorylated in quiescent cells, but was phosphorylated on serine and to a lesser extent threonine, several hours following cell stimulation. Furthermore, annexin VI also incorporated phosphate in a growth-dependent manner, in a form other than a phosphoamino-acid. The phosphate was visualised following acid hydrolysis of immunoprecipitated annexin VI, as part of a complex having high mobility on 2-D thin-layer electrophoresis. The identity of this complex is not known. The results suggest that a post-translational modification other than direct protein phosphorylation may influence the activity of annexin VI and provide evidence linking cell growth with regulation of annexin VI function.

Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities.

Many DNA helicases utilise the energy derived from nucleoside triphosphate hydrolysis to fuel their actions as molecular motors in a variety of biological processes. In association with RuvA, the E. coli RuvB protein (a hexameric ring helicase), promotes the branch migration of Holliday junctions during genetic recombination and DNA repair. To analyse the relationship between ATP-dependent DNA helicase activity and branch migration, a site-directed mutation was introduced into the helicase II motif of RuvB. Over-expression of RuvBD113N in wild-type E. coli resulted in a dominant negative UVs phenotype. The biochemical properties of RuvBD113N were examined and compared with wild-type RuvB in vitro. The single amino acid substitution resulted in major alterations to the biochemical activities of RuvB, such that RuvBD113N was defective in DNA binding and ATP hydrolysis, while retaining the ability to form hexameric rings and interact with RuvA. RuvBD113N formed heterohexamers with wild-type RuvB, and could inhibit RuvB function by affecting its ability to bind DNA. However, heterohexamers exhibited an ability to promote branch migration in vitro indicating that not all subunits of the ring need to be catalytically competent.

Activation of protein kinase C modulates the expression of the T3/T cell antigen receptor complex on human T lymphocytes.

Activators of protein kinase C induced a rapid decrease (within 15 min) in the surface expression of the T3 antigen and T-lymphocyte antigen receptor (Ti) on HPB-ALL cells, and a concomitant phosphorylation of the T3 gamma and delta polypeptides; the gamma chain was more extensively phosphorylated than the delta chain. No phosphorylation of the T3 epsilon chain and the Ti alpha and beta polypeptides was detected. Evidence was obtained that the T3 gamma chain is phosphorylated only on serine residues.

Identification of calcium-binding proteins associated with the lymphocyte plasma membrane.

The Nonidet P40 insoluble fraction of lymphocyte plasma membrane contains three polypeptides of about 68,000-, 33,000- and 28,000-Mr which are solubilised by Ca2+-chelators. As judged by various criteria the 33,000-Mr polypeptide is homologous to the 36,000-Mr pp60src kinase substrate of chicken fibroblasts and the 68,000-Mr polypeptide is related to the 67,000-Mr "calelectrin" of bovine liver. The 28,000-Mr polypeptide may also be related to calelectrin.

Activators of protein kinase C down-regulate and phosphorylate the T3/T-cell antigen receptor complex of human T lymphocytes.

As judged by indirect immunofluorescence, phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol induced a rapid, concentration-dependent decrease of about 50% in the surface expression of the T3 antigen on human T lymphoblasts, and of T3 and the T-cell antigen receptor on HPB-ALL cells. Direct binding experiments using 125I-labeled antibody indicated that the reduction in T3 expression corresponded to a decrease in the number of antigen molecules rather than a change in their affinity. Biochemical analyses revealed that phorbol dibutyrate induced a rapid, prominent phosphorylation of the T3 Mr 26,000 gamma chain and to a lesser extent of the Mr 21,000 delta chain. No phosphorylation of the T3 epsilon chain or of the alpha and beta subunits of the T-cell antigen receptor was detected. The data suggest that protein kinase C induces a phosphorylation of the T3 gamma and delta chains that may lead to the down-regulation of the T3/T-cell antigen receptor complex.

Branch migration and Holliday junction resolution catalyzed by activities from mammalian cells.

During homologous recombination, DNA strand exchange leads to Holliday junction formation. The movement, or branch migration, of this junction along DNA extends the length of the heteroduplex joint. In prokaryotes, branch migration and Holliday junction resolution are catalyzed by the RuvA and RuvB proteins, which form a complex with RuvC resolvase to form a "resolvasome". Mammalian cell-free extracts have now been fractionated to reveal analogous activities. An ATP-dependent branch migration activity, which migrates junctions through >2700 bp, cofractionates with the Holliday junction resolvase during several chromatographic steps. Together, the two activities promote concerted branch migration/resolution reactions similar to those catalyzed by E. coli RuvABC, highlighting the preservation of this essential pathway in recombination and DNA repair from prokaryotes to mammals.

CD5 is phosphorylated on tyrosine after stimulation of the T-cell antigen receptor complex.

When T cells are activated by the T-cell antigen receptor, a number of cellular proteins are phosphorylated on tyrosine. We investigated whether any of these proteins were present on the surface of activated T cells. Using the human leukemic T-cell line Jurkat and normal peripheral blood lymphocytes, we identified a 67-kDa cell surface glycoprotein in anti-phosphotyrosine immunoprecipitates, after treatment of the cells with CD3 antibody. When cell lysates were depleted of CD5 by sequential immunoprecipitation, the 67-kDa phosphotyrosyl polypeptide was no longer precipitated by the phosphotyrosine antibody. Western blot analysis of anti-phosphotyrosine precipitates confirmed that this glycoprotein was CD5. It was possible that CD5 was present in the anti-phosphotyrosine immunoprecipitates due to its physical association with phosphotyrosyl proteins rather than being directly tyrosine-phosphorylated itself. However, Western blot analysis of anti-CD5 immunoprecipitates with phosphotyrosine antibody and phosphoamino acid analysis demonstrated that CD5 was indeed phosphorylated on tyrosine after stimulation of the cells with CD3 antibody and was concomitantly phosphorylated on serine and threonine. Tyrosine phosphorylation of CD5 was maximal 2 min after CD3 stimulation and returned to baseline levels by 60 min. CD5 is expressed on the cell surface of all mature T cells and a small proportion of B lymphocytes and has recently been identified as the ligand for CD72, a receptor present on the surface of all B cells. The present data suggest that tyrosine phosphorylation may be involved in B-cell-T-cell communication.

Evidence that a GTP binding protein regulates phosphorylation of the CD3 antigen in human T lymphocytes.

The role of guanine nucleotide binding regulatory proteins (G proteins) in the regulation of phosphorylation of the gamma subunit of the CD3 antigen has been examined. CD3 gamma chain phosphorylation in isolated T cell microsomes was stimulated by the G protein activator guanosine 5'-0 thiotriphosphate (GTP gamma S), but cyclic adenosine monophosphate and guanosine 5'-diphosphate were ineffective at inducing gamma chain phosphorylation. The effect of GTP gamma S was rapid and transient; a half maximal effect was observed with 50 microM of the nucleotide. gamma polypeptide phosphorylated in vitro in GTP gamma S stimulated microsomes incorporated phosphate on Serines 123 and 126. These data are consistent with the involvement of a G protein in the signalling mechanisms that regulate the phosphorylation of the CD3 gamma chain.

Evidence that a kinase distinct from protein kinase C induces CD3 gamma-subunit phosphorylation without a concomitant down-regulation in CD3 antigen expression.

An immediate consequence of Ag-specific activation of T cells is phosphorylation of the gamma-subunit of the CD3 gamma-chain. There is good evidence that the kinase that mediates CD3 gamma-chain phosphorylation is protein kinase C (pkC). It has also been proposed that the interaction between pkC and CD3 gamma-chains controls the cell surface expression of the antigen receptor/CD3 Ag complex. In the present study we present data relevant to these two points. Thus we show that CD3 gamma-subunit phosphorylation can be triggered by the calcium ionophore ionomycin. However, as judged by several criteria, ionomycin does not stimulate cellular pkC. Accordingly, ionomycin must regulate phosphorylation of the CD3 Ag by a kinase distinct from pkC. The phosphorylation of CD3 Ag induced by ionomycin is not accompanied by a modulation of the cell surface expression of CD3 molecules which implies that CD3 gamma-chain phosphorylation is not a sufficient signal for the endocytosis of the CD3/Ag receptor complex.

The T cell receptor/CD3 complex and CD2 stimulate the tyrosine phosphorylation of indistinguishable patterns of polypeptides in the human T leukemic cell line Jurkat.

Stimulation of the T cell receptor (TcR)/CD3 complex of Jurkat T cells with a monoclonal antibody to the CD3 epsilon chain induced the tyrosine phosphorylation of multiple polypeptides, ranging in size from 21 to 155 kDa. The protein tyrosine phosphorylation was characterized by its rapidity and its transient nature, returning to baseline levels by 60 min. Protein tyrosine kinase activity was also induced when the Jurkat T cells were stimulated with a mitogenic pair of antibodies directed against CD2. Comparison of the polypeptides which were phosphorylated on tyrosine in response to stimulation of the two receptors, by either one- or two-dimensional analysis, failed to reveal any differences. These data suggest that the TcR/CD3 complex and CD2 activated the same tyrosine kinase or kinases. A model is proposed in which CD2 functions as a signal amplifier in physiological responses to antigen/major histocompatibility complex without changing the qualitative nature of the signal generated via the TcR/CD3 complex.

Association of phosphorylation of the T3 antigen with immune activation of T lymphocytes.

In human T lymphocytes the antigen receptor (Ti) is associated non-covalently on the cell surface with the invariant T3 antigen which comprises 3 chains: two glycosylated polypeptides of relative molecular mass 26,000 (Mr 26K) and 21K (gamma and delta) and one non-N-glycosylated polypeptide of Mr 19K (epsilon). The proposed function of T3 is to transduce the activation signals delivered via the antigen receptor. Recently we have shown that phorbol esters, which stimulate protein kinase C, can induce phosphorylation of the gamma subunit of the T3 antigen. But the critical question is whether T3 phosphorylation occurs as a normal consequence of immune activation of T lymphocytes. In this respect, it has been shown that immune stimulation of murine T cells results in phosphorylation of Ti-associated polypeptides that may be the functional analogues of the human T3 antigen. We have therefore monitored T3 phosphorylation after exposure of human T cells to antigen or phytohaemagglutinin (PHA). The data show that both stimuli initiate phosphorylation of the gamma subunit of the T3 antigen which indicates that T3 phosphorylation is a physiological response to immune activation.

Resolution of recombination intermediates by a mammalian activity functionally analogous to Escherichia coli RuvC resolvase.

A mammalian endonuclease that resolves Holliday junctions has been partially purified from extracts of calf thymus and Chinese hamster ovary cells. The activity acts upon (i) synthetic Holliday junctions and (ii) recombination intermediates made by the Escherichia coli RecA protein and appears to be functionally analogous to the E. coli RuvC protein. Cleavage occurs by the introduction of symmetrically related nicks in strands of like polarity to produce nicked duplex DNA products. The nicks can be repaired by DNA ligase. The resolvase is specific for Holliday junctions and does not act upon Y junctions, G/A mismatches, or heterologous loops. The substrate specificity is therefore similar to that of E. coli RuvC protein and contrasts with the broad range specificity of other junction resolvases such as T4 endonuclease VII. The mammalian resolvase activity has been observed at normal levels in extracts prepared from a series of DNA repair-defective cells. These include the x-ray or UV-sensitive hamster lines xrs-5, xrs-6, and Chinese hamster ovary 43-3B (defective in ERCC-1), and murine cells that are severely immunodeficient and defective in both V(D)J rejoining and DNA repair.

The cell surface and its metabolism.

The cell surface structure is highly dynamic. In particular, binding of ligand induces the redistribution of receptors on the cell surface as well as the internalisation of ligand-receptor complexes. Internalisation in turn leads to a recycling of the receptor or to a decrease in the cell's responsiveness to the ligand. Modulation of the cell surface structure is apparently regulated intracellularly by components of the cell's cytoskeleton. A crucial component in this respect is likely to be a sub-membranous filamentous network that is linked directly to the cytoplasmic face of the surface membrane. In erythrocytes this network can be separated from purified preparations of the plasma membrane by virtue of its insolubility in nonionic detergents. Application of this procedure to the plasma membrane fraction of human B lymphoblastoid cells has yielded a detergent-insoluble residue comprising actin and a 68,000-Mr polypeptide as major components, together with polypeptides of 28,000-, 33,000- and 120,000-Mr as prominent but more minor components. The association of the 68,000-Mr protein with the detergent-insoluble residue and the original plasma membrane is Ca2+-dependent. Burkitt lymphoma cells differ noticeably from lymphoblastoid cells in that the 68,000-Mr protein is not associated with the inner face of the surface membrane. This difference may reflect the malignant phenotype of Burkitt lymphomas or the hypothetical sub-population of normal B lymphocytes from which the lymphomas are derived.

Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue.

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5'-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

Characterization of distinct tyrosine-specific protein kinases in B and T lymphocytes.

Lymphocyte membrane fractions from both normal and neoplastic sources exhibit tyrosine-specific protein kinase activity. The molecular weights of the endogenous substrates phosphorylated on tyrosine residues differ in B and T cells. To further characterize membrane tyrosine phosphorylation in the two major classes of lymphocytes, the tryptic phosphopeptides of their endogenous substrates were compared and the sensitivity of the kinases to inhibition by N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) was determined. The two major B cell substrates (61,000 and 55,000 daltons, p61 and p55) were gel purified after phosphorylation and exhaustively digested with trypsin. Separation by reverse phase high pressure liquid chromatography demonstrated that these two substrates had two identical phosphotyrosine containing tryptic phosphopeptides. p61 had an additional phosphotyrosine site. Parallel analysis of the two T cell substrates (64,000 and 58,000 daltons, p64 and p58) showed that they also contained two phosphotyrosine sites that were identical. However, the tryptic phosphopeptides from the B and T cell substrate pairs were clearly distinct suggesting that they arise from different gene products. When B and T cell membrane fractions were preincubated with TLCK (21 degrees C, 30 min) a dose-dependent decrease in p64 and p58 phosphorylation resulted. p61 and p55 phosphorylation was not affected at concentrations up to 10 mM TLCK. Tyrosine-specific kinase activity was also assessed by measuring phosphorylation of a tyrosine containing synthetic peptide. The kinase activity of T cell plasma membrane fractions was inhibited by TLCK; the B cell activity was unaffected. The results suggest that membrane fractions from normal and some neoplastic B and T cells have at least two different tyrosine-specific kinases.

The human T3 gamma chain is phosphorylated at serine 126 in response to T lymphocyte activation.

The gamma subunit of the human T lymphocyte T3 antigen is rapidly phosphorylated on serine residues in vivo during the initiation of T cell activation by a polyclonal mitogen (Phaseolus vulgaris phytohemagglutinin), an activator of protein kinase C (phorbol 12,13-dibutyrate), and an elevator of intracellular calcium (ionomycin). The sites of phosphorylation were identified by comparing tryptic peptide analyses of T3 gamma chains labeled in vivo with various synthetic peptides, corresponding to portions of the cytoplasmic domain of the gamma chain that had been labeled in vitro using purified protein kinase C. Two sites, serines 123 and 126, were phosphorylated in response to ionomycin, whereas a single site, serine 126, was phosphorylated when T lymphocytes were stimulated by P. vulgaris phytohemagglutinin or when protein kinase C was directly activated by phorbol 12,13-dibutyrate. Immune activation of T cells via the protein kinase C pathway thus induces phosphorylation of a single site on the T3 gamma chain, namely serine 126.