Requirement for sialic acid on the endothelial ligand of a lymphocyte homing receptor

The entry of blood-borne lymphocytes into most secondary lymphoid organs is initiated by a highly specific adhesive interaction with the specialized cuboidal endothelial cells of high endothelial venules (HEV). The adhesive receptors on lymphocytes that dictate interactions with HEV in different lymphoid organs are called homing receptors, signifying their critical role in controlling organ-selective lymphocyte migration. Considerable work has established that the mouse peripheral lymph node homing receptor (pnHR), defined by the mAb MEL- 14, functions as a lectin-like adhesive protein. We have previously shown that sialidase treatment of peripheral lymph node (PN) HEV abrogates lymphocyte attachment to the HEV both in vivo and in vitro. We extend this evidence by demonstrating that Limax agglutinin (LA), a sialic acid-specific lectin, when reacted with HEV exposed in cryostat- cut tissue sections, blocks lymphocyte attachment to PN HEV and, unexpectedly, to the HEV of Peyer's patches (PP) as well. Using a recombinant form of the pnHR as a histochemical probe for its cognate adhesive site (HEV-ligand) on PN HEV, we demonstrate that both sialidase and Limax agglutinin functionally inactive this ligand. It is concluded that the requirement for sialic acid is at the level of the pnHR interaction with its HEV ligand. A distinct sialyloligosaccharide may encode the recognition determinant of a PP HEV ligand.     

Receptors involved in lymphocyte homing: relationship between a carbohydrate-binding receptor and the MEL-14 antigen

Blood-borne lymphocytes extravasate in large numbers within peripheral lymph nodes (PN) and other secondary lymphoid organs. It has been proposed that the initiation of extravasation is based upon a family of cell adhesion molecules (homing receptors) that mediate lymphocyte attachment to specialized high endothelial venules (HEV) within the lymphoid tissues. A putative homing receptor has been identified by the monoclonal antibody, MEL-14, which recognizes an 80-90-kD glycoprotein on the surface of mouse lymphocytes and blocks the attachment of lymphocytes to PN HEV. In a companion study we characterize a carbohydrate-binding receptor on the surface of mouse lymphocytes that also appears to be involved in the interaction of lymphocytes with PN HEV. This receptor selectively binds to fluorescent beads derivatized with PPME, a polysaccharide rich in mannose-6-phosphate. In this report we examine the relationship between this carbohydrate-binding receptor and the putative homing receptor identified by the MEL-14 antibody. We found that: MEL-14 completely and selectively blocks the activity of the carbohydrate-binding receptor on mouse lymphocytes; the ability of six lymphoma cell lines to bind PPME beads correlates with cell-surface expression of the MEL-14 antigen, as well as PN HEV-binding activity; selection of lymphoma cell line variants for PPME-bead binding by fluorescence-activated cell sorting (FACS) produces highly correlated (r = 0.974, P less than 0.001) and selective changes in MEL-14 antigen expression. These results show that the carbohydrate-binding receptor on lymphocytes and the MEL-14 antigen, which have been independently implicated as receptors involved in PN-specific HEV attachment, are very closely related, if not identical, molecules.     

Phosphomannosyl-derivatized beads detect a receptor involved in lymphocyte homing

Recirculating lymphocytes initiate extravasation from the blood stream by binding to specialized high endothelial venules (HEV) within peripheral lymph nodes (PN) and other secondary lymphoid organs. We have previously reported that lymphocyte attachment to PN HEV is selectively inhibited by mannose-6-phosphate (M6P) and related carbohydrates (Stoolman, L. M., T. S. Tenforde, and S. D. Rosen, 1984, J. Cell Biol., 99:1535-1540). In the present study, we employ a novel cell-surface probe consisting of fluorescent beads derivatized with PPME, a M6P-rich polysaccharide. PPME beads directly identify a carbohydrate-binding receptor on the surface of mouse lymphocytes. In every way examined, lymphocyte attachment to PPME beads (measured by flow cytofluorometry) mimics the interaction of lymphocytes with PN HEV (measured in the Stamper-Woodruff in vitro assay): both interactions are selectively inhibited by the same panel of structurally related carbohydrates, are calcium-dependent, and are sensitive to mild treatment of the lymphocytes with trypsin. In addition, thymocytes and a thymic lymphoma, S49, bind poorly to PPME beads in correspondence to their weak ability to bind to HEV. When the S49 cell line was subjected to a selection procedure with PPME beads, the ability of the cells to bind PPME beads, as well as their ability to bind to PN HEV, increased six- to eightfold. We conclude that a carbohydrate-binding receptor on mouse lymphocytes, detected by PPME beads, is involved in lymphocyte attachment to PN HEV.     

Intravenously injected sialidase inactivates attachment sites for lymphocytes on high endothelial venules

Blood-borne lymphocytes initiate entry into secondary lymphoid organs, such as peripheral lymph nodes (PN) and gut-associated Peyer's patches (PP), by a highly specific adhesive interaction between the lymphocytes and the endothelium of specialized blood vessels known as a high endothelial venules (HEV). The selectivity with which functional subpopulations of lymphocytes migrate into particular lymphoid organs is believed to be regulated by the expression of cell adhesion receptors and complementary ligands on lymphocytes and HEV, respectively. The entry of lymphocytes into PN and PP has clearly been shown to involve distinct receptor-ligand pairs. Employing the Stamper-Woodruff in vitro adhesion assay, which measures lymphocyte attachment to HEV in cryostat-cut sections of lymphoid organs, we have previously shown that treatment of PN sections with two different sialidases inactivates HEV-adhesive ligands, whereas treatment of PP tissue sections has no effect on HEV-adhesive function. We now report that in vivo exposure of HEV to sialidase (after i.v. injection of the enzyme) also selectively prevents subsequent in vitro attachment of lymphocytes to PN HEV but not to PP HEV. Consistent with this organ-selective impairment of HEV-adhesive function by sialidase, i.v. injection of the enzyme is shown to prevent short term lymphocyte accumulation within peripheral lymph nodes while having no significant effect on accumulation in PP, blood, or nonlymphoid organs. Histologic examination with the sialic acid-specific lectin from Limax flavus verified that i.v. injected sialidase effectively removes stainable sialic acid moieties from HEV in both PN and PP. This study confirms that sialic acid is required for the adhesive function of PN HEV-ligands. A role for sialic acid as either a recognition determinant or as a regulatory molecule can be envisioned. In view of the fact that many pathogens release sialidase and cause substantially elevated serum levels of this enzyme, the present observations may have pathophysiologic significance. One mechanism by which such pathogens may avoid destruction is to inactivate susceptible HEV-ligands and disrupt the entry of lymphocytes into lymphoid organs where immune responses against the pathogens would normally be initiated.     

2′(3′),5′-ADP inhibits initiation-dependent protein synthesis in a cell-free system from Ehrlich ascites tumor cells

When tested in a poly(U)-dependent polyphenylalanine synthesizing system and in a postnuclear supernatant, both derived from Ehrlich ascites tumor cells, 2(3),5-ADP did not affect chain elongation of polypeptide synthesis. In a cell-free system which was dependent on initiation and programmed by natural mRNA, however, the amino acid incorporating activity was suppressed to about 10% of the control in the presence of 1 mM 2(3),5-ADP. The inhibitor was shown not to interfere with the attachment of poly(U) to the small ribosomal subunit and with the formation of mRNA-80S ribosome complexes in a complete protein synthesizing system. The subsequent attachment of a 40S ribosomal subunit to the mRNA-80S ribosome complex and the formation of polysomes, however, was depressed by the inhibitor. The experimental results suggest that 2(3),5-ADP inhibits initiation-dependent protein synthesis between monosome formation and the formation of the first peptide bond(s).     

Tetramethyl Rhodamine Methyl Ester (TMRM) is Suitable for Cytofluorometric Measurements of Mitochondrial Membrane Potential in Cells Treated with Digitonin

A new method for cytofluorometric analysis of mitochondrial membrane potential has been developed by using TMRM as a cationic, mitochondrial selective probe. The method is based on limited treatment of cultured cells with digitonin which permeabilises the plasma membrane and leaves mitochondria intact. The resulting signal of TMRM-stained cells thus represents only the probe accumulated in mitochondria. Fibroblasts and cybrids were used as a model cell systems and optimal conditions for digitonin treatment and staining by TMRM were described. The TMRM signal collapsed by valinomycin, KCN and antimycin A and FCCP titration was used to gradually lower and characterise the stability of . The method is suitable for sensitive measurement of in different types of cultured cells.     

Demonstration that a lectin-like receptor (gp90MEL) directly mediates adhesion of lymphocytes to high endothelial venules of lymph nodes

Lymphocyte migration from the blood into most secondary lymphoid organs is initiated by a highly selective adhesive interaction with the endothelium of specialized blood vessels known as high endothelial venules (HEV). The propensity of lymphocytes to migrate to particular lymphoid organs is known as lymphocyte homing, and the receptors on lymphocytes that dictate interactions with HEV at particular anatomical sites are designated "homing receptors". Based upon antibody blockade experiments and cell-type distribution studies, a prominent candidate for the peripheral lymph node homing receptor in mouse is the approximately 90-kD cell surface glycoprotein (gp90MEL) recognized by the monoclonal antibody MEL-14. Previous work, including sequencing of a cDNA encoding for this molecule, supports the possibility that gp90MEL is a calcium-dependent lectin-like receptor. Here, we show that immunoaffinity-purified gp90MEL interacts in a sugar-inhibitable manner with sites on peripheral lymph node HEV and prevents attachment of lymphocytes. Lymphocyte attachment to HEV in Peyer's patches, a gut-associated lymphoid organ, is not affected by gp90MEL. The results demonstrate that gp90MEL, as a lectin-like receptor, directly bridges lymphocytes to the endothelium.     

Lymphocyte adhesion to cultured Peyer's patch high endothelial venule cells is mediated by organ-specific homing receptors and can be regulated by cytokines

Adhesion of lymphocytes to high endothelial venule (HEV) cells is the first step in the migration of these cells from blood into lymph nodes and Peyer's patches (PP). In the present study, we isolated and cultured HEV cells from PP of the rat and assessed their capacity to interact with lymphocytes. Flow cytometric analysis with a rat HEV-specific mAb KJ-4 revealed that greater than 90% of the cultured cells were stained by the antibody. Furthermore, confluent monolayers of PP HEV cells retained the capacity to support the adhesion of lymphocytes from spleen, thoracic duct, and lymph nodes but not binding of immature cells from thymus and bone marrow, which are deficient in cells capable of binding to HEV in vivo. In addition, intraepithelial lymphocytes that preferentially migrated into mucosal lymphoid tissues were also enriched in cells that adhered to the endothelial monolayers. The binding process required energy, was calcium-dependent, and could be inhibited by cytochalasin D, trypsin, and mixed glycosidase. Interestingly, pretreatment of PP HEV cells with rTNF, IFN-gamma, or granulocyte-macrophage CSF significantly increased the endothelial adhesiveness for thoracic duct lymphocytes in a time- and dose-dependent manner. In contrast, stimulation of lymphocytes with phorbol ester or TNF resulted in the rapid modulation of the surface expression of the PP homing receptor and decrease in lymphocyte binding to normal or TNF-stimulated HEV cells. The adhesion of lymphocytes to normal or cytokine-stimulated HEV cells can be blocked by pretreatment of lymphocytes, but not HEV cells, with the PP homing receptor-specific 1B.2.6 antibody. Taken together, these experiments provide strong evidence that the interaction between lymphocytes and cultured HEV cells are mediated by adhesive mechanisms that regulate lymphocyte entry into PP in vivo and that cytokines can promote HEV adhesiveness for lymphocytes through increased expression of organ-specific ligands on HEV cells.     

Characterization of a cDNA encoding the 55 kDa B regulatory subunit of Arabidopsis protein phosphatase 2A

Type 2A serine/threonine protein phosphatases (PP2A) are key components in the regulation of signal transduction and control of cell metabolism. The activity of these protein phosphatases is modulated by regulatory subunits. While PP2A activity has been characterized in plants, little is known about its regulation. We used the polymerase chain reaction to amplify a segment of a cDNA encoding the B regulatory subunit of PP2A from Arabidopsis. The amplified DNA fragment of 372 nucleotides was used as a probe to screen an Arabidopsis cDNA library and a full-length clone (AtB) of 2.1 kbp was isolated. The predicted protein encoded by AtB is 43 to 46% identical and 53 to 56% similar to its yeast and mammalian counterparts, and contains three unique regions of amino acid insertions not present in the animal B regulatory subunit. Genomic Southern blots indicate the Arabidopsis genome contains at least two genes encoding the B regulatory subunit. In addition, other plant species also contain DNA sequences homologous to the B regulatory subunit, indicating that regulation of PP2A activity by the 55 kDa B regulatory subunit is probably ubiquitous in plants. Northern blots indicate the AtB mRNA accumulates in all Arabidopsis tissues examined, suggesting the protein product of the AtB gene performs a basic housekeeping function in plant cells.     

Interleukin-6 involvement in mesothelioma pathobiology: inhibition by interferon α immunotherapy

A role for interleukin-6 (IL-6) in malignant mesothelioma has been suggested by the clinically presenting symptoms of mesothelioma patients, which include fever, weight loss and thrombocytosis. A murine model of malignant mesothelioma was therefore used to examine the potential role of IL-6 in this cancer type and whether the effect of interferon (IFN) therapy on mesothelioma might be mediated, in part, by regulating IL-6 levels and/or IL-6-induced pathobiology. A panel of human and murine mesothelioma cell lines was assayed for endogenous IL-6 production in a bioassay, and for IL-6-mRNA expression. Four out of 5 human and 5 out of 15 murine mesothelioma cell lines produced moderate to high levels of bioactive IL-6 in vitro. This result was corroborated by mRNA detection. One of the representative murine cell lines, AB22, was chosen for further in vivo studies in the murine mesothelioma model. In AB22-inoculated mice detectable serum IL-6 levels were found to precede macroscopically detectable tumour growth, clinical signs (cachexia, abdominal distension, diarrhoea) and changes in the peripheral lymphoid organs (cell depletion and functional depression). Treatment with anti-IL-6 antibody curtailed the clinical symptoms (P<0.001), as did treatment with recombinant human (rhu) IFN (P<0.001). Neither anti-IL-6 antibody nor rhuIFN had a direct growth-inhibitory effect on the AB22 mesothelioma cell line in vitro, however, in vivo rhuIFN treatment of mice inoculated with AB22 cells attenuated both IL-6 mRNA expression in the tumours and serum IL-6 levels, ameliorated the depression of lymphocyte activities, and enhanced the number of tumour-infiltrating lymphocytes and macrophages. On the basis of these results it is suggested that IL-6 mediates some of these effects, directly or indirectly, and that a combination therapy of rhuIFN and anti-IL-6 antibody may be an improved palliative treatment for patients with malignant mesothelioma.     

Induction of interleukin-2 receptor by tumor necrosis factor α on cultured ovarian tumor-associated lymphocytes

Summary We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor (TNF) in the induction, expansion, long-term proliferation and lytic function of CD8⁺ TAL. TNF up-regulated the IL-2 receptor (IL-2R) chain (Tac antigen) on the surface of CD3⁺ CD8⁺ CD4^– TAL, enhanced the proliferation of autologous tumor-specific CTL, and potentiated their lytic function in long-term cultures. Furthermore, in the induction and expansion phase of CD8⁺ TAL, the presence of TNF was associated with a selective increase in CD8⁺ IL-2R⁺ (Tac⁺) cells, and subsequent decrease in CD4⁺ IL-2R⁺ (Tac⁺) cells. These results suggest that the observed facilitation of the outgrowth of CD8⁺ cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNF in the analysis of signalling in autologous tumor-reactive CTL.     

Characterization and gene organization of Taiwan banded krait (Bungarus multicinctus) gamma-bungarotoxin

-Bungarotoxin was isolated from Bungarus multicinctus (Taiwan banded krait) venom using a combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase high-performance liquid chromatography column. Circular dichroism (CD) measurement revealed that its secondary structure was dominant with -sheet structure as is that of snake venom -neurotoxins and cardiotoxins. -Bungarotoxin exhibits activity on inhibiting the binding of [³H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine receptor subtype, and competes weakly with radioiodinated -bungarotoxin for binding to the Torpedo nicotinic acetylcholine receptor. Moreover, the toxin inhibits collagen-induced platelet aggregation, with an IC₅₀ of approximately 200 nM. The genomic DNA encoding the -bungarotoxin precursor is amplified by polymerase chain reaction (PCR). The gene is organized with three exons separated by two introns, and shares virtually identical overall organization with those reported for -neurotoxin and cardiotoxin genes, including similar intron insertions. The intron sequences of these genes share sequence identity up to 85%, but the exon sequences are highly variable. These observations suggest that -bungarotoxin, -neurotoxins, and cardiotoxins originate from a common ancestor, and the evolution of these genes shows a tendency to diversify the functions of snake venom proteins.     

Directional components derived from initial-orientation data of inexperienced homing pigeons

Summary 1,324 vanishing bearings of individual pigeons, completely inexperienced in homing, were recorded during 100 releases at 36 sites symmetrically distributed around their home loft near Würzburg at distances ranging from 7 to 180 km. Two directional components could be derived from the data: (a) a weak but significant homeward component pointing to the loft site and (b) a more pronounced compass component pointing to the northwest (Fig. 1). With the latter kind of tendency the pigeons clearly demonstrated what earlier had been described as preferred compass direction (PCD).Homeward directedness at the 36 differently situated release sites is negatively correlated with angular divergence between PCD and homeward direction (Fig. 2). Compass preferences derived from different quartets of symmetrically arranged sites were similar. Over a period of 11 years the PCD varied significantly in time but did not change fundamentally (Figs. 3, 4). Interrelations between various parameters of initial-orientation data were investigated by correlation analyses (Table 3).PCDs have not only been found in the Würzburg area but around many other loft sites as well. They are, however, differently pronounced, and in experienced pigeons they are generally weaker than in first-flight birds. Various reasons (partly evident, partly thought possible) for variability of PCDs, and for their absence in some series of experiments, are discussed.As a PCD persists, and even tends to become clearer, after elimination of homeward orientation by olfactory deprivation, it is concluded that it reflects directional tendencies which are independent of the process of site localization. A great deal of what is called release-site biases, i.e., site-specific deviations from the beeline course towards home, can be understood on a PCD basis. The functional background of the PCD is unknown. Hypothetical advantages for the pigeons homing strategy are considered.Abbreviations RSB release-site bias - PCD preferred compass direction     

The effect of humidity and light on cellular water relations and diffusion conductance of leaves ofTradescantia virginiana L.

Turgor (_p) and osmotic potential (_s) in epidermal and mesophyll cells, in-situ xylem water potential (-xyl) and gas exchange were measured during changes of air humidity and light in leaves ofTradescantia virginiana L., Turgor of single cells was determined using the pressure probe. Sap of individual cells was collected with the probe for measuring the freezing-point depression in a nanoliter osmometer. Turgor pressure was by 0.2 to 0.4 MPa larger in mesophyll cells than in epidermal cells. A water-potential gradient, which was dependent on the rate of transpiration, was found between epidermis and mesophyll and between tip and base of the test leaf. Step changes of humidity or light resulted in changes of epidermal and mesophyll turgor (_p-epi, _p-mes) and could be correlated with the transpiration rate. Osmotic potential was not affected by a step change of humidity or light. For the humidity-step experiments, stomatal conductance (g) increased with increasing epidermal turgor.g/_p-epi appeared to be constant over a wide range of epidermal turgor pressures. In light-step experiments this type of response was not found and stomatal conductance could increase while epidermal turgor decreased.Symbols E transpiration - g leaf conductance - w leaf/air vapour concentration difference - -epi water potential of epidermal cells - -mes water potential of mesophyll cells - -xyl water potential of xylem - _p-epi turgor pressure of epidermal cells - _p-mes turgor pressure of mesophyll cells - _s-epi osmotic potential of epidermal cells - _s-mes osmotic potential of mesophyll cells     

Specialized and generalized dispersal systems: where does ‘the paradigm’ stand?

I explore the specialization versus generalization paradigm in frugivory and seed dispersal. This view predicts that some tropical trees produce nutritious fruits adapted for use by a small coterie of specialized frugivores that provide reliable seed dissemination. Other tree species are expected to offer superabundant fruits of lower nutritional reward, relying instead on common opportunistic frugivores that are individually less reliable, but collectively disperse seeds effectively. Though widely referenced, many aspects of the paradigm are untested with tropical trees and avian frugivores, primarily because plant ecologists rarely determine whether specialist or generalist foragers are responsible for different patterns of seed distribution, while students of foraging behavior rarely determine the effects seed dispersal by different animals for survival of seeds or seedlings of specialist or generalist trees.Ecological paradigms provide alternative hypotheses, without evolutionary arguments. Keystone species have ecological effects disproportionate to their abundance; it is important for management considerations to know whether fruiting trees or frugivores serve as keystone mutualists in tropical forests. Alternatively, the extent to which vertebrate seed dispersers influence density-dependent seed, seedling, sapling, or adult mortality may have important consequences for spatial dispersion and population dynamics of tree species in tropical forests.     

Ischemic preconditioning ameliorates microcirculatory disturbance through downregulation of TNF-alpha production in a rat cremaster muscle model

Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, and the accumulation and infiltration of neutrophils and macrophages, which disturbs the microcirculatory hemodynamics. Nonetheless, ischemic preconditioning (IPC) is known to produce immediate tolerance to subsequent prolonged I/R insults, although its underlying mechanism largely remains unknown. Our study investigated the role of the IB--NF-B-TNF- (tumor necrosis factor-) pathway in IPC's ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps. Male Sprague-Dawley rats were randomized (n=8 per group) into 3 groups: a sham-operated control group, an I/R group (4 h of pudic epigastric artery ischemia followed by 2 h of reperfusion), and an IPC+I/R group (3 cycles of 10 min of ischemia followed by 10 min reperfusion before I/R). Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in cremaster muscles. I/R markedly increased the number of rolling, adhering, and migrating leukocytes. It was also observed that I/R significantly increased TNF- expression in these injured tissues. On the other hand, IPC prevented I/R-induced increases in leukocyte rolling, adhesion, and transmigration. Moreover, TNF- protein production and its mRNA expression were downregulated in the IPC group. Finally, I/R-induced IB- phosphorylation and NF-B (p65) nuclear translocation were both suppressed by IPC. These results indicated that IPC attenuated NF-B activation and subsequently reduced TNF- expression, which resulted in the amelioration of microcirculatory disturbances in I/R-injured cremaster muscles.     

Endogenous abscisic acid and 2-trans-abscisic acid in alternate bearing ‘Wilking’ mandarin trees

The relationship of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) to alternate bearing has been examined in Wilking mandarin (Citrus reticulata Blanco) trees. Leaves, stems and buds of trees loaded with fruit (on trees) had 4.3, 6.0 and 2.2 fold higher ABA levels than the corresponding organs from off trees. Leaves had higher ABA levels than stems and buds in both on and off trees. t-ABA was non-detectable in Wilking leaf, stem and bud tissue. Amounts of t-ABA not exceeding 40% of the ABA content, were found in Shamouti and Valencia orange buds and in Wilking fruit peel.The elevated levels of ABA in on tree organs may reflect a stress imposed by the fruit overload.     

Transformation of 16-dehydroprogesterone and 17α-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17-hydroxyprogesterone (II, 17-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7, 14-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Ic), and 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14-position (Ia) followed by hydroxylation at the 7-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17, 20-dihydroxypregn-4-en-3-one (IIa), 7, 17-dihydroxypregn-4-ene-3, 20-dione (IIb), 6, 17, 20-trihydroxypregn-4-en-3-one (IIc) and 11, 17, 20-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 or 11 position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.     

γδ T-cell receptor repertoire in human peripheral blood and thymus

T-cell clones expressing the T-cell receptor (Tcr) were generated from peripheral blood lymphocytes (PBLs) and from a thymus sample. In the panel of ten thymus-derived clones, four Tcr phenotypes [as defined by the reaction of monoclonal antibodies (mAbs) directed against known V and V regions] were identified. All the clones lacked expression of the V3 V region, while seven clones were V1+ . V1 was found in combination with V9 or with undefined VVregions. In addition, two other Tcr phenotypes were identified on these clones: V9⁺ V1^– V3^– and V9^– V1^– V3^– One of the clones expressed CD4 and another was CD8positive. The remaining clones were CD4^– CD8^–. In the panel of 76 PBL-derived, Tcr-bearing clones, five Tcr phenotypes could be identified. In contrast to the thymus-derived clones, 30% of the clones were V3⁺ whereas V1 was expressed by a minority of the clones only. One clone was CD4-positive and approximately 30% of the clones were CD8-positive. Four of the five mAb-defined Tcr phenotypes could be identified on both thymus and PBL-derived T-cell clones. However, biochemical analysis of the Tcrs demonstrates differences in the usage of Ct- and C2-encoded y chains by T cells derived from the thymus and PBLs. The results therefore indicate that, at the clonal level, similarities and differences exist between the Tcr repertoires expressed in the thymus and by PBLs. Furthermore, they indicate that combinatorial Tcr heterogeneity is larger than has so far been described. The receptor diversity, combined with the potential of Tcr+ cells to express CD4 or CD8, indicates that these cells are a heterogeneous population that might mediate a number of immune functions.     

5-Nucleotidase activity in lymphocytes

Summary Some characteristics of lymphocyte 5-nucleotidase are reviewed. The optimal conditions for the cytochemical localization of 5-nucleotidase (AMPase) in the mouse lymphocyte have been established. Quantitative monitoring of the effects of fixation and the components of the cytochemical medium showed that cytochemistry can be performed under conditions that do not lead to loss of AMPase activity. The cytochemical reaction product was seen only on the surface of a proportion of splenic lymphocytes, regardless of the fixative used. The splenic cell population included a distinct population of lymphocytes with readily demonstrable AMPase activity and another population with no cytochemically demonstrable AMPase activity. The number of positive cells varied, but was usually between 20 and 30% when cells were fixed in glutaraldehyde. Lymphocytes purified from thymus were always negative for cytochemically demonstrable AMPase activity. Biochemically, it was shown that the AMPase activity of spleen lymphocytes is more than six times higher than that of thymus lymphocytes. 5-Nucleotidase activity in normal and abnormal lymphocytes is discussed in the light of the latest findings. The possible function of 5-nucleotidase in lymphocytes is discussed.