Cytolytic T cells activated by H-2-controlled E molecules cross-react with A molecules

Cell-mediated lymphocytotoxicity was generated in four strain combinations differing only by the cell-surface expression of the class II E molecule controlled by the H-2 complex. The four combinations were: B10.D2(R107) anti-B10.A(3R), B10.A(4R) anti-B10.A(2R), B10.GD anti-B10.D2(R101), and B10.S(7R) anti-B10.S(9R). In all four of these combinations, the stimulator expresses E molecules on the cell surface, while the responder does not. The cytolytic T lymphocytes generated in the B10.D2(R107) anti-B10.A(3R) and B10.A(4R) anti-B10.A(2R) combinations reacted not only with the stimulator but also with strains that do not express cell-surface E molecules, in particular, strains carrying the H-2 ^f and H-2 ^q haplotypes. The cross-reactivity with E-negative strains could be blocked by monoclonal antibodies specific for the A^f or A^q molecules but not by antibodies recognizing determinants on E or class I (K) molecules. The anti-H-2^f cross-reactivity could be inhibited by H-2 ^q cold targets and, reciprocally, the anti-H-2^q reactivity could be blocked by H-2 ^f cold targets. These findings are interpreted as indicating that the cytolytic T lymphocytes stimulated by E molecules can recognize and lyse cells lacking E molecules but expressing A molecules. The observed E-A cross-reactivity supports the notion of structural and functional relatedness between the A and E molecules and suggests a common evolutionary origin of the A- and E-encoding loci.     

Abnormal blood group galactosyltransferase in blood type A1B-subjects whose sera contain anti-B agglutinin

Summary A blood type A₁B female, whose plasma agglutinates B red blood cells from other subjects but not her own, was found. Her sister with blood type A₁B (sister-1) exhibited the same peculiarity. The agglutination titer of red cells from these two subjects is lower than that of normal B. Her father is blood type B, and his plasma did not agglutinate B red cells from other subjects and his own. Her mother and another sister (sister-2) were usual blood type A₁. Blood group galactosyltransferase (B-enzyme) activity of plasma from the propositus, sister-1, and their father was very low. Km for 2-fucosyllactose of B-enzyme of these subjects was 1.3 mM, which was more than two times higher than that of the normal value. Km for UDP-Gal was similar, but the pH-activity profile differed for the two enzymes.Red cell membranes from her father contained about 70% ungalactosylated H-sites, whereas, virtually all H-sites are galactosylated in the usual B red cell membranes. The blood group ABH components are known to be heterogeneous. Because of the abnormal B-enzyme with low activity and low substrate affinity, some H components might not be galactosylated, particularly in A₁B cases (i.e., the propositus and her sister-1), due to competition by A₁ enzyme. The lack of certain B components is likely to be the cause of the existence of the anti-B agglutinin in their sera.     

Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands

Summary Cytochemical localization of blood group ABH antigens was examined in secretory cells of human cervical glands by application of a post-embedding lectin-gold as well as immuno-gold labeling procedure using monoclonal antibodies. Blood group specific lectins such as Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Griffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) and Ulex europaeus agglutinin-I (UEA-I) reacted with secretory granules but not with other cytoplasmic organellae such as nucleus and cell membrane. The reactivity of secretory granules with these lectins showed strict dependence on the blood group and secretor status of tissue donors. The binding patterns with these lectins were not homogeneous, but exhibited marked cellular and subcellular heterogeneity. Thus, for example, in blood group A individuals, some granules were stained strongly with DBA and others were weakly or not at all with the lectin. Such a heterogenous labeling with the lectin was observed even in the same cells. Similar results were obtained with UEA-I and GSAI-B₄ staining in blood group O and B secretor individuals, respectively. Monoclonal antibodies likewise reacted specifically with the granules but they occasionally bound to some nucleus. The labeling pattern of the antibodies with the granules was essentially the same as those of lectins. However, difference was also observed between monoclonal antibody and lectin staining, that is, monoclonal anti-A antibody reacted weakly but consistently with granules from blood group A nonsecretors but DBA (HPA) did not; staining with UEA-I was observed in granules from the secretor individuals of any blood groups whereas monoclonal anti-H antibody reacted with granules from blood group O and some A secretor individuals but not from B and AB secretor individuals; GSAI-B₄ reacted uniformly with granules throughout the cells whereas monoclonal anti-B antibody bound to limited number of granules in the same cells. This was confirmed by the double labeling experiments with the lectin and the antibody. These results suggest that the different types of antigens as to the binding ability for monoclonal antibodies and lectins are expressed on different granules in the same cell.     

Cocaine produces D2R-mediated conformational changes in the adenosine A2AR-dopamine D2R heteromer

Adenosine A_2A receptors (A_2ARs) and dopamine D₂ receptors (D₂Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A_2ARs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A_2AR and D_2LR formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D₂R homodimers and in A_2AR-D₂R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time- and concentration-dependent reduction in the BRET_max between A_2AR-D_2LR heterodimers and D_2LR homodimers, but not A_2AR homodimers, indicating its effect on D₂R. Cocaine was evaluated with regard to D₂R binding using a human D_2LR stable expressing CHO cell line and was found to produce an increase in the affinity of hD_2LR for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPγS. However, cocaine failed to modulate D₂R agonist-induced inhibition of cAMP in stable hD_2LR CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D_2LR, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D₂R homodimers and A_2AR-D₂R heterodimers.     

Interaction of pneumococcal S-14 polysaccharide with lectins from Ricinus communis,Triticum vulgaris, and bandeiraea simplicifolia

Two purified lectins, namely, wheat-germ agglutinin (from Triticum vulgaris) and the hemagglutinin from Ricinus communis seeds, readily form a precipitate with pneumococcal S-14 polysaccharide, whereas the Bandeiraea simplicifolia lectin (BS 1) does not. Exhaustive periodate oxidation and borohydride reduction of S 14 modifies terminal β-D-galactopyranosyl residues, as well as chain D-glucopyranosyl residues, and abolishes reactivity with both the R. communis lectin and wheat-germ agglutinin. Controlled periodate oxidation followed by Smith degradation cleaves only terminal β-D-galactopyranosyl residues, giving a linear polymer, the structure of which was determined by methylation analysis. This derived polymer, containing (1→6)-linked 2-acetamido-2-deoxy-β-D-glucosyl residues, readily precipitated wheat-germ agglutinin, but not the R. communis lectin.     

Endothelin-1/Endothelin A receptor-mediated biased signaling is a new player in modulating human ovarian cancer cell tumorigenesis

The endothelin-1 (ET-1)/endothelin A receptor (ET_AR, a G protein-coupled receptor) axis confers pleiotropic effects on both tumor cells and the tumor microenvironment, modulating chemo-resistance and other tumor-associated processes by activating Gαq- and β-arrestin-mediated pathways. While the precise mechanisms by which these effects occur remain to be elucidated, interference with ET_AR signaling has emerged as a promising antitumor strategy in many cancers including ovarian cancer (OC). However, current clinical approaches using ET_AR antagonists in the absence of a detailed knowledge of downstream signaling have resulted in multiple adverse side effects and limited therapeutic efficacy. To maximize the safety and efficacy of ET_AR-targeted OC therapy, we investigated the role of other G protein subunits such as Gα_s in the ET_AR-mediated ovarian oncogenic signaling. In HEY (human metastatic OC) cells where the ET-1/ET_AR axis is well-characterized, Gαs signaling inhibits ET_AR-mediated OC cell migration, wound healing, proliferation and colony formation on soft agar while inducing OC cell apoptosis. Mechanistically, ET-1/ET_AR is coupled to Gαs/cAMP signaling in the same ovarian carcinoma-derived cell line. Gαs/cAMP/PKA activation inhibits ET_AR-mediated β-arrestin activation of angiogenic/metastatic Calcrl and Icam2 expression. Consistent with our findings, Gαs overexpression is associated with improved survival in OC patients in the analysis of the Cancer Genome Atlas data. In conclusion, our results indicate a novel function for Gαs signaling in ET-1/ET_AR-mediated OC oncogenesis and may provide a rationale for a biased signaling mechanism, which selectively activates Gαs-coupled tumor suppressive pathways while blocking Gαq-/β-arrestin-mediated oncogenic pathways, to improve the targeting of the ET_AR axis in OC.     

Role of c-Cbl carboxyl terminus in serotonin 5-HT2A receptor recycling and resensitization

The 5-hydroxytryptamine 2A receptor (5-HT_2AR) undergoes constitutive and agonist-dependent internalization. Despite many advances in our understanding of G protein-coupled receptor trafficking, the exact mechanism of endocytic sorting of G protein-coupled receptors remains obscure. Recently, we have reported a novel finding documenting a global role for the ubiquitin ligase c-Cbl in regulating vesicular sorting of epidermal growth factor receptor (Baldys, A., Göoz, M., Morinelli, T. A., Lee, M. H., Raymond, J. R., Jr., Luttrell, L. M., and Raymond, J. R., Sr. (2009) Biochemistry 48, 1462–1473). Thus, we tested the hypothesis that c-Cbl might play a role in 5-HT_2AR recycling. In this study, we demonstrated an association of 5-HT_2AR with c-Cbl. Furthermore, down-regulation of c-Cbl by RNA interference blocked efficient recycling of 5-HT_2AR to the plasma membrane. Immunofluorescence microscopy revealed that 5-HT_2A receptors were trapped in early endosome antigen 1- and Rab11-positive sorting endosomes in cells overexpressing c-Cbl mutants lacking carboxyl termini. This inhibitory effect was associated with a relative decrease in association of c-Cbl truncation proteins with the 5-HT_2AR, compared with that observed for the full-length c-Cbl fusion protein. Consistent with the delayed recycling, 5-HT_2AR resensitization was greatly attenuated in the presence of c-Cbl mutants lacking carboxyl termini, as detected by changes in the cytosolic calcium. Taken together, these studies have led to the discovery that the C-terminal region of c-Cbl plays a crucial role in the temporal and spatial control of 5-HT_2AR recycling.     

Expression of the I-E target antigen for T-cell killing requires two genes

TheH?2I ^k region encodes at least two different target antigens for unrestricted T-cell mediated killing. The first is controlled by theI?A region alone and the second depends on a pair of alleles, one located to the left ofI?B (presumably inI?A) and the other to the right ofI?J (presumably inI?E). Hence, effector cells nominally specific for a product of theI?E region do not kill target cells with the sameI?E region as the stimulator unless theI?A region is also shared. Some effectors specific forH?2I ^k , such as A.TH anti-A.TL and B10.A(4R) anti-B10.A(2R), cross-react with B10.A(3R) and B10.A(5R) target cells. A product of theH?2 ^b haplotype was shown to complement products of theH?2 ^d orH?2 ^k haplotypes in forming this cross-reactive determinant. The results are consistent with recent biochemical data on the component chains of Ia antigens.     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

Specific activation of human T lymphocytes by Robinia pseudoacacia seeds' lectin.

Human peripheral blood and tonsil lymphocytes were fractionated into T and B cells by centrifugation after rosetting with native sheep erythrocytes and tested with Robinia pseudoacacia lectin. The purity of B- and T-enriched populations was checked by E-rosette formation or heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to Robinia lectin from all the donors tested was not found to differ from that of unfractionated cells, whereas no response of highly purified B cells could be observed to the lectin even with different concentrations of the lectin and different culture periods. B cells, however, were found to bind as much ³H labeled Robinia lectin as unfractionated lymphocytes. In addition, treatment of cells by antihuman T-lymphocyte antigen (HTLA) serum and complement before addition of Robinia lectin completely abolished their response, whereas similar treatment by antihuman B lymphocyte and monocyte antigen (HBLMA) serum did not prevent the T cells from incorporating thymidine. The Robinia lectin, like the other phytomitogens, thus appears to be a specific T-cell activator.     

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A_2A receptor (A_2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A_2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A_2AR C-terminus from 412 to 316 amino acids (A_2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A_2AR levels. As cAMP production is downstream of the first activation event—coupling of G protein to its receptor—investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gα_s proteins, we characterize the association of A_2AR and A_2AΔ316R to Gα_s with and without GDP or GTPγs using surface plasmon resonance (SPR). Gα_s affinity for A_2AR was greatest for apo-Gα_s, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A_2AR C-terminus (A_2AΔ316R) decreased the affinity of the unliganded receptor for Gα_s by ～20%, suggesting small changes to binding can greatly impact downstream signaling.     

Distribution of Bandeiraea simplicifolia lectin binding sites in the genital organs of female and male mice

Summary Fluorescein isothiocyanate labelled type I lectin from Bandeiraea simplicifolia (BSA-I) known for its specific binding to -d-galactopyranosyl and 2-acetamido-2-deoxy-d-galactose groups, has been used to map the distribution of the lectin specific binding sites in the genital organs of female and male mice. In non-pregnant female mice, strong lectin reactivity was restricted to the epithelium of the distal oviduct, the cervix and vagina. In pregnant mice strong BSA-I reactivity was also noted in the epithelium of uterine glands from the time of implantation on day 5 onward. In the testis BSA-I bound selectively to sperm but did not react with other cells in the seminiferous tubules. In the proximal caput epididymis BSA-I reacted with the epithelial cells, the underlying basement membranes and the intraluminal sperm. The intraluminal contents of the seminal vesicles reacted strongly with the lectin. Our data thus show a widespread but selective distribution of BSA-I lectin binding sites in the male and female genital organs and altered lectin binding in the uterus during pregnancy.     

Schinus terebinthifolius Leaf Extract Causes Midgut Damage,Interfering with Survival and Development of Aedes aegypti Larvae

In this study, a leaf extract from Schinus terebinthifolius was evaluated for effects on survival, development, and midgut of A. aegypti fourth instar larvae (L4), as well as for toxic effect on Artemia salina. Leaf extract was obtained using 0.15 M NaCl and evaluated for phytochemical composition and lectin activity. Early L4 larvae were incubated with the extract (0.3–1.35%, w/v) for 8 days, in presence or absence of food. Polymeric proanthocyanidins, hydrolysable tannins, heterosid and aglycone flavonoids, cinnamic acid derivatives, traces of steroids, and lectin activity were detected in the extract, which killed the larvae at an LC₅₀ of 0.62% (unfed larvae) and 1.03% (fed larvae). Further, the larvae incubated with the extract reacted by eliminating the gut content. No larvae reached the pupal stage in treatments at concentrations between 0.5% and 1.35%, while in the control (fed larvae), 61.7% of individuals emerged as adults. The extract (1.0%) promoted intense disorganization of larval midgut epithelium, including deformation and hypertrophy of cells, disruption of microvilli, and vacuolization of cytoplasms, affecting digestive, enteroendocrine, regenerative, and proliferating cells. In addition, cells with fragmented DNA were observed. Separation of extract components by solid phase extraction revealed that cinnamic acid derivatives and flavonoids are involved in larvicidal effect of the extract, being the first most efficient in a short time after larvae treatment. The lectin present in the extract was isolated, but did not show deleterious effects on larvae. The extract and cinnamic acid derivatives were toxic to A. salina nauplii, while the flavonoids showed low toxicity. S. terebinthifolius leaf extract caused damage to the midgut of A. aegypti larvae, interfering with survival and development. The larvicidal effect of the extract can be attributed to cinnamic acid derivatives and flavonoids. The data obtained using A. salina indicates that caution should be used when employing this extract as a larvicidal agent.     

Studium der schwachen B-Variante in einer Familie

Zusammenfassung Es wird eine Familie beschrieben, in der bei 17 von 31 untersuchten Mitgliedern eine schwache B-Variante vorkommt (B-Grouppe bei 13, AB-Gruppe bei 4 Personen). Die serologischen Eigenschaften des schwachen B-Antigens, ebenso der Prozentsatz der mit Anti-B nichtagglutinierten Erythrocyten, unterschieden sich von Fall zu Fall, so daß die Klassifizierung der B-Variante bei einzelnen Personen unterschiedlich war. Alle Träger des schwachen B-Antigens sind Nichtausscheider der B-Substanzen und Ausscheider von H(A). Bei 2 Familienmitgliedern mit der A- und AB-Blutgruppe wurde eine Abnormalität der phänotypischen Ausprägung des A-Antigens festgestellt; die A_HP-Antigene zeigen eine normale Expressivität. Diese Ergebnisse sprechen für die Unabhängigkeit der A- und A_HP-Gene. Die Rolle der modifizierenden Gene yy für das Auftreten von A- und B-Antigenen an Erythrocyten und im Speichel wird diskutiert.

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Study of the weak B variant in a family

Summary A family is described, in which a weak B variant occurred in 17 from 31 examined members (B group in 13 cases, AB group in 4 persons). Both the serological properties of the weak B antigen and the percent of the red cells non-agglutinated with anti-B differed as the case may be; the classification of the weak B antigen varied therefore in single persons. All the carriers of the weak B were non-secretors of B and secretors of H(A). By 2 members of the family of the A and AB group an abnormality concerning the phenotype expression of the A antigen was found, whereas the A_HP receptors had a normal antigenicity. This result proves an independence of the A and A_HP genes. The role of the suppression genes yy for the phenotypic expresion of the A and B antigens in the red cells and in the saliva is discussed.

     

An endothelin type A receptor-expressing cell to characterize endothelin-1 binding and screen antagonist

Endothelin-1 (ET-1) induces contraction of vascular smooth muscle through binding to endothelin type A receptor (ET_AR). COS-7 cells stably expressing high levels of the ET_AR were established (designated COS-7(ET_AR)). The COS-7(ET_AR) cell bound [¹²⁵I]ET-1 with a K_d of 932 ± 161 pM and a B_max of 74 ± 13 fmol/2 × 10⁵ cells. [¹²⁵I]ET-1 binding was inhibited by ET-1 and the ET_AR antagonist BQ-610, but not by the endothelin type B receptor (ET_BR) antagonist BQ-788. In clones expressing two ET_AR mutants containing D46N or R53Q substitutions in the first extracellular domain of the receptor, [¹²⁵I]ET-1 binding activity was dramatically reduced. This suggests that these single amino acid substitutions alter the three-dimensional structure of the ligand-binding domain of the ET_AR. Using COS-7(ET_AR) cell, we showed that Ca²⁺ or Mg²⁺ was essential for ET-1 binding to the ET_AR and that ET-1 treatment induced postreceptor signaling, that is, intracellular accumulation of cyclic AMP (cAMP) and Ca²⁺ mobilization. The COS-7(ET_AR) established in this study will be a useful tool for screening ET-1 antagonists for treating hypertension.     

Effect of progesterone administration on the distribution of oviductal carbohydrates in <Emphasis Type="Italic">Rana tigrina</Emphasis>

Our aim has been to determine whether carbohydrate distribution in the oviducts of progesterone-treated animals is comparable with that of seasonal breeders in Rana tigrina. Like many other anurans, R. tigrina oviduct exhibits a short straight portion (pars recta, pr) at the beginning followed by a long, highly coiled portion (pars convoluta, pc). Histologically, the oviduct of this species revealed some unique features, one of which was intense toluidine blue staining, specifically in the upper mucosal glands of pc4. Based on lectin reactivities in the epithelial cells and mucosal glands, patterns of lectin staining in the seasonal breeders were classified into seven types: R1-R3 (for pr) and C1-C4 (for pc). Typically, some lectins reacted selectively either with ciliated cells (concanavalin A) or non-cialiated cells (Ricinus communis agglutinin I and wheatgerm agglutinin); however, Bandeiraea simplicifolia agglutinin I reacted with both cell types. These staining patterns were different in the progesterone-treated animals. Differences in glycan distribution in the oviductal secretions were revealed by lectin blotting. Compared with the seasonal breeders, an enhanced staining of some lectins was noted in the hormone-treated animals: either an increased staining intensity of existing protein bands or additional staining of new protein bands. Inversely, the staining of wheatgerm agglutinin was markedly diminished in the hormone-treated animals, suggesting the inhibitory effect of progesterone on oviductal glycan distribution. Whether alteration in glycan distribution upon progesterone treatment affects the physiological properties of the released jelly substances remains to be addressed. This research was supported by Thailand Research Funds (to W.W.), a Research Initiate Grant from Kasetsart University (to A.T.), and Mahidol University.     

Novel 8-(furan-2-yl)-3-substituted thiazolo [5,4-e][1,2,4] triazolo[1,5-c] pyrimidine-2(3H)-thione derivatives as potential adenosine A2A receptor antagonists

Novel thiazolotriazolopyrimidine derivatives (23–33) designed as potential adenosine A_2A receptor (A_2AR) antagonists were synthesized. Molecular docking studies revealed that all compounds (23–33) exhibited strong interaction with A_2AR. The strong interaction of the compounds (23–33) with A_2AR in silico was confirmed by their high binding affinity with human A_2AR stably expressed in HEK293 cells using radioligand-binding assay. The compounds 24–26 demonstrated substantial binding affinity and selectivity for A_2AR as compared to SCH58261, a standard A_2AR antagonist. Decrease in A_2AR-coupled release of endogenous cAMP in treated HEK293 cells demonstrated in vitro A_2AR antagonist potential of the compounds 24–26. Attenuation in haloperidol-induced motor impairments (catalepsy and akinesia) in Swiss albino male mice pre-treated with compounds 24–26 further supports their role in the alleviation of PD symptoms.     

DIFFERENTIAL LOCALIZATION OF CELL SURFACE AND SECRETORY COMPONENTS IN RAT INTESTINAL EPITHELIUM BY USE OF LECTINS

Sections through various levels of small intestine from adult male rats were examined by fluorescence microscopy after treatment with fluorescein isothiocyanate-labeled lectins from Dolichos biflorus, Lotus tetragonolobus, Ricinus communis, and Triticum vulgare (wheat germ). The latter three lectins reacted with the microvillar portion of the epithelial cells lining the crypts and villi in sections of intestine adjacent to the pylorus. This pattern of reactivity was sharply altered along the first 15 cm of intestine so that in sections distal to this point the luminal surfaces of only those epithelial cells in the crypts and at the base of the villi reacted with the L. tetragonolobus and R. communis lectins, whereas the wheat germ lectin reacted with the surfaces of the cells lining the villi. In sections from the distal end of the small intestine, all three lectins reacted with the surfaces of cells only at the base of the villi and in the crypts. These results show a difference in surface components in cells at various portions on the villi and the dependence of these differences on the region of intestine. The D. biflorus lectin reacted with approximately 25% of the goblet cells at each level of intestine studied whereas the reactivities of the goblet cells with the other three lectins were dependent upon the region of intestine.     

Babesia bovis: Proteins of virulent and avirulent parasites passaged through ticks and splenectomized or intact calves

Passage of the avirulent vaccine (K) strain of Babesia bovis (K_A) through either Boophilus microplus ticks, intact calves, or intact calves and then ticks, resulted in two distinct protein and protein antigen profiles as analyzed by two-dimensional gel electrophoresis of biosynthetically labeled proteins and immunoprecipitates. Different degrees of expression of two major acidic antigens of K_A designated Ka₁ (M_r 47,500) and Ka₂ (M_r 43,000) were observed. Ka₁ was apparently lost following passage of K_AB. bovis through intact calves but was strongly represented in the parasite population following a single tick passage. In contrast, passage through ticks of the virulent K_VB. bovis (from which K_A was derived by passage in splenectomized calves) did not lead to strong representation of the Ka₁ protein although there was increased representation of another major acidic protein antigen, designated Kv (M_r 35,000). These data suggest that the previously recognized reversion to a strain-dependent basal antigenic type in the tick vector depends also on intrastrain characteristics such as virulence and strain heterogeneity. The data suggest that K_A is a more heterogeneous population than K_V although cloned isolates are required to establish this point. Comparable syringe passage of another strain of B. bovis, designated C strain, through splenectomized calves resulted in less marked differences between the putative C_A and C_VB. bovis. This may explain the less stable avirulence of C_A compared to K_AB. bovis. Various selection pressures must act, in either the tick or the vertebrate host, on subpopulations in heterogeneous isolates to produce the changes described in protein antigen profiles of B. bovis. The possible relevance of changes in representation of proteins to biological characteristics of B. bovis (such as virulence and tick transmissibility) is discussed.     

Complexity of lectins from the hard roe of perch (Perca fluviatilis L.)

Fish eggs are a rich source of lectins, the sugar-binding (glyco)proteins. In this paper we aim to further characterise perch roe lectins using several protein characterisation techniques including affinity chromatography and protein sequencing. Perch roe lectins are comprised of two subunits, subunit A and subunit B which have molecular weights of 12,400 and 12,000, respectively. These subunits form multiple aggregates A_nB_n in which the two subunits are present in differing ratios and, also as an `homogeneous' aggregates of one of the subunits A_n or B_n. Lectins A_n (designated A thereafter) and lectin B_n (designated B thereafter) formed by one type of subunit only (subunit A or B) were isolated in a pure state. Lectin B could also be isolated by spontaneous precipitation occurring during incubation of the perch roe extract at 4°C. Lectin B has a higher affinity for d-glucose than lectin A, whereas both lectins (A and B) have a similar affinity for l-fucose. The N-terminal region of subunit B showed the following amino acid sequence: EPAXPPWGTQFG-, whereas the N-terminus of subunit A was blocked and therefore could not be directly sequenced. Differences between subunits A and B were also found in amino acid composition. This unusual complexity and variability of perch roe lectins is likely to have physiological significance which, as yet, remains to be determined.