Agglutinability of cattle red cells. 4. The effect of antiglobulin in comparison with treatment by pronase

The average titre scores varied from zero to 24.7 for the cattle red cells (CRC) from different MZ pairs. These CRC were sensitized with different blood-typing reagents and were titre-tested against the double dilution series of an anti-bovine y-globulin serum. A significant negative correlation (r = - 0.82; P <0.001) was found between the degree of agglutinability and the amount of neuraminic acid of the surface component of CRC cleaved by pronase.
After pronase treatment of the CRC it could be demonstrated that (1) the activity of the V and E'₃ blood factors became destroyed; (2) three new specific receptors became evolved; (3) the degree of 'direct' agglutinability due to the A₂, O₃ W, S₂ and Z anti-sera did not parallel with the titre scores obtained in the anti-globulin tests.     

Agglutinability of cattle red cells. 1. Agglutination by lectins of enzyme-treated red cells

Pronase-treated cattle red cells (CRC) from different monozygous (MZ) twin pairs could be classified as weakly (titre 1: 2, 1: 4) or strongly (titre 1: 1024, 1: 4096) agglutinable when Phytohemagglutinin-M (Phy-M) was used as agglutinin. In the presence of concavalin-A (Con-A), the CRC from different MZ pairs treated with pronase, A-chymotrypsin or trypsin showed a gradation from low (titre 1: 32) to high (titre 1: 256 000) agglutinability. The trypsin-treated CRC which had A₁, A₂ blood factors usually had a titre of 1: 8000 or higher with Con-A. Both the intact CRC and the CRC treated with proteolytic enzymes were capable of absorbing the Phy-M or Con-A lectins.     

Agglutinability of cattle red cells. 3. Conglutination.

Addition of both complement and conglutinin was necessary to conglutinate the cattle red cells (CRC) when they were sensitized by different blood typing reagents. Although the degree of conglutinability of the CRC was influenced by the particular blood factor-reagent combination the average conglutinability (i.e. average titre scores) of CRC from different MZ pairs varied from 1.9 to 16.2. The titres of complement varied from zero to 1:32, while the titres of conglutinin ranged from 1:8 to 1:1024 in the different sera from MZ cattle twins. The variance due to differences in the titre scores between MZ pairs was 82.2% for conglutinin and 68.3% for complement. There was no evident association between the titres of conglutinin and complement.     

Agglutinability of cattle red cells. 3. Conlutination

Addition of both complement and conglutinin was necessary to conglutinate the cattle red cells (CRC) when they were sensitized by different blood typing reagents. Although the degree of conglutinability of the CRC was influenced by the particular blood factor-reagent combination the average conglutinability (i.e. average titre scores) of CRC from different MZ pairs varied from 1.9 to 16.2.
The titres of complement varied from zero to 1:32, while the litres of conglutinin ranged from 1:8 to 1:1024 in the different sera from MZ cattle twins. The variance due to differences in the titre scores between MZ pairs was 82.2 % for conglutinin and 68.3 % for complement. There was no evident association between the titres of conglutinin and complement.     

CONCANAVALIN A-INDUCED AGGLUTINATION AND BINDING OF CON A TO THE DIFFERENTIATING CELLS OF DICTYOSTELIUM DISCOIDEUM

Changes in agglutinability of Dictyostelium discoideum cells with Concanavalin A (Con A) during the course of development were investigated. The agglutinability of the cells was assayed under conditions where no spontaneous cell agglutination occurred. It was found that there was a progressive decrease in Con A-induced agglutinability during development: a decrease to half from exponentially growing cells to preaggregation cells, and to sixth in disaggregated slug cells. Pronase-BAL treatment of preaggregation cells did not enhance their agglutinability with Con A. The amounts of sites available for binding Con A were determined with preaggregation and slug cells. Cells were incubated at 4°C and in the presence of NaN₃ to avoid possible endocytosis of Con A. No significant differences in numbers of Con A-binding sites per unit area of cell surface was detected among preaggregation cells, those treated with pronase and BAL and cells disaggregated from slugs by similar treatment. It was thus concluded that the decrease in Con A-induced agglutinability during development is not attributable to changes in the numbers of Con A-binding sites.     

Agglutinability of cattle red cells. 2. Agglutination by isologous and heterologous sera of enzyme-treated red cells

Neuraminidase treatment made cattle red cells (CRC) agglutinable by the agglutinins of the different heterologous but not of the homologous sera. These agglutinins were, however, absorbed by both the nauraminidase-treated and the intact CRC.
Proteolytic treatment made CRC agglutinable also by the normal cattle, isoimmune and autologous sera. Agglutination titres of the CRC ranged from 1: 2 to 1: 256, but the variation between CRC from members of monozygous (MZ) pairs was not greater than ± 2 agglutination score units the range of experimental error.
Treatment with trypsin made the A₁, A₂ factors more emergent on the surface of CRC for agglutination by anti-A₂, while pronase treatment had a similar effect upon agglutination of Z-positive cells by anti-Z.     

Alterations in reactivity of the blood factors of cattle red cells after pronase treatment.

Pronase treatment of cattle red cells produced various effects: (a) an increase in reactivity of the J factor and evolution of a specific cryptoantigen; (b) decrease in the A-B, G2, K, I1, O2, O3, P, Q, T1, Y2, A', B', E'2, E'3, I', K', O'-L'-V-L and M2 factors, but (c) no change in the specifity or in the titre of the remaining 16 different blood factors. Most of the pronase-affectable blood factors were destroyed in a rather narrow but characteristic range of pronase treatment intensities. However, at like intensities, variations were demonstrable due to the fact that the blood factor occurred (a) in red cells from different individuals, and (b) in different phenogroups or subgroups of the B locus.     

Mating reaction inSaccharomyces cerevisiae

The effect of proteolytic enzymes on sexual agglutinability of haploid cells of the yeastSaccharomyces cerevisiae was examined. Sexual agglutinability of cells of botha and α types was lost on treatment with alkaline protease and two kinds of neutral proteases ofBacillus subtilis, pronase and α-chymotrypsin. Agglutinability of α type cells was lost after treatment with acid protease ofRhizopus chinensis and trypsin, but that ofa type cells was not. These results indicate that the sex-specific substance responsible for the sexual agglutination (agglutination factor) ina type cells differs from that in α type cells. Agglutination factors were solubilized from cell-wall fractions of both mating types by Glusulase treatment. These crude factors specifically inhibited the agglutinability of cells of the opposite mating type with little effect on the agglutinability of cells of the same mating type.     

Concanavalin A-mediated agglutination of 3T3 cells after exposure to UV-irradiated herpes simplex virus.

Studies were made to determine the effect of UV-irradiation of herpes simplex virus (HSV) on Concanavalin A (Con-A)-mediated agglutination of 3T3 cells. There were three different phases of agglutination by Con-A of cells infected with HSV. The agglutinability began to increase from 3 or 4 hr, or 72 hr after exposure of cells to HSV. The early-appearing agglutinability was further divided into two phases, based on its sensitivity to metabolic inhibitors. These were tentatively called "Early 1 or inhibitor sensitive", "Early 2 or inhibitor insensitive" and "Late" agglutinability. "Early 1" agglutination, detected from 3 hr post infection (pi), was induced by treating cells with HSV, either active or UV-irradiated for less than 5 min and was inhibited when actinomycin D (1 microgram/ml) or cycloheximide (50 microgram/ml) was added to the cultures. "Early 2" agglutination began to increase from 4 hr pi when cells were inoculated with HSV irradiated for 7 to 20 min and was not affected by either inhibitor. HSV irradiated for 6 min failed to induce either agglutinability. "Late" agglutination, observed 72 hr pi, was detected in cultures which had been treated with HSV irradiated for 4 to 15 min. Among those, virus irradiated for 6 to 8 min was most efficient. HSV-transformed cells were also agglutinated without exception by low concentrations of Con-A.     

Alterations in reactivity of the blood factors of cattle red cells after pronase treatment

Pronase treatment of cattle red cells produced various effects: (a) an increase in reactivity of the J factor and evolution of a specific cryptoantigen; (b) decrease in the A-B, G₂, K, I₂ O₂, O₃, P, Q, T_1; Y₂, A, B', E'₂, E'₃, I', K', O'-L'-V-L and M, factors, but (c) no change in the specifity or in the titre of the remaining 16 different blood factors. Most of the pronase-affectable blood factors were destroyed in a rather narrow but characteristic range of pronase treatment intensities. However, at like intensities, variations were demonstrable due to the fact that the blood factor occurred (a) in red cells from different individuals, and (b) in different phenogroups or subgroups of the B locus.     

The serodiagnosis of human hydatid disease: 1. The routine use of latex-agglutination and complement-fixation in diagnosis

The combined use of complement fixation (CF) and latex agglutination (LA) tests is reported on sera from 6328 patients with suspected hydatid disease; 191 were confirmed positive at operation ('known positives'). Results by LA are related to CF titres. Both tests were negative in 90% of specimens. Nine patients were subsequently found infected of whom 3 became positive in tests after operation. Of sera positive in both tests, 75% were from 'known positives'. The remainder were almost certainly from infected patients. Half the patients whose sera were LA positive/CF less than or equal to 1/4 were follow-up 'known positives' in whom CF titres had waned; 2 were early infections. Only 3% of the cases with an LA negative/CF titre of greater than or equal to 1/16 were 'known positives' and 6% where the CF titre was 1/8. The remaining CF results in the group were false positives and accounted for 1.2% of all sera tested. Findings show that a CF titre greater than or equal to 1/8 with positive LA indicates past or present infection; a negative CF test with positive LA usually indicates past infection; rarely, infection is present when a CF titre is greater than or equal to 1/8 and LA is negative. A rising CF titre and positive LA indicates current infection; reliable prognosis following treatment is given by CF.     

Differential lectin-mediated agglutinabilities of the embryonic and the first extraembryonic cell line of the early chick embryo

Summary The lectin-mediated agglutinability of cells dissociated from different areas of the gastrulating chick embryo was investigated. Differences in agglutinability were quantified by using a Coulter counter. Cells from the area pellucida (AP) and those from the endoderm of the area opaca (AOEn) are agglutinated by Concanavalin A (Con A), wheat germ agglutinin (WGA) andRicinus communis agglutinin (RCA). In cells from both areas the greatest agglutination response is obtained with RCA. Trypsinization of AOEn cells enhances their agglutinability with Con A, WGA and RCA. The lectin-induced agglutinability of cells from the area pellucida is similar in EDTA-dissociated and trypsinized cells.Cells from the AP are significantly more agglutinable with Con A than those of the AOEn regardless whether the former are obtained by trypsinization or dissociation with EDTA. The higher agglutinability of cells of the area pellucida with Con A, as well as the differential enhancement by trypsin of the agglutinability of AOEn cells with Con A, WGA, and RCA may reflect a difference in the cell surface glycoreceptors between the cells of the are pellucida (predominantly embryonic) and the first extraembryonic (AOEn) cell line. These cells have been shown to sort out from each other at the earliest stages of development.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes.

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and nonagglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. This was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination. Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg2+, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca2+ (0.2 mM) had little effect on agglutinability, although high Ca2+ (5 mM) inhibited agglutinability of the resealed membranes somewhat. Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells. The agglutination of erythrocytes was not affected by cytochalasin B (40 mug/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes. It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca2+ or Mg2+ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitivie to vinblastine.     

Lack of correlation between agglutinability,the surface distribution of con A and post-confluence inhibition of cell division in ten cell lines

Agglutinability by concanavalin A, distribution of surface-bound concanavalin A, and maximal cell density in monolayer culture were examined under similar conditions in parallel cultures of ten established cell lines. The degree of agglutinability of the cell lines did not correlate with the presence or absence of patching of concanavalin A bound to the cell surface, as determined with a hemocyanin marker. Agglutinability was also not always correlated with the loss of post-confluence inhibition of cell division. Two clones of mouse 3T3 fibroblasts that maintained post-confluence inhibition of cell division and low agglutinability differed substantially with respect to the surface distribution of concanavalin A. Patching of concanavalin A binding sites is neither necessary nor sufficient to explain differences in agglutinability between cell lines.     

An enzyme basis for blood type A intermediate status.

The blood type A is known to be subclassified as A1, A2, and A1-A2 intermediate (Aint), depending upon red cell agglutinability with anti-A1 and anti-H lectins. Approximately 80% of the blood group H-sites remained unglycosylated in type Aint erythrocyte membranes. Plasma from Aint individuals contains a special blood group GalNAc transferase (UDP-GalNAc:2''-fucosylgalactoside-alpha-3-N-acetylgalactosaminyl transferase), which is different from the enzyme in A1 plasma and the enzyme in A2 plasma. A1-enzyme has strong affinity to UDP-GalNAc and 2''-fucosyllactose, A2-enzyme has low affinity to both substrates, and Aint-enzyme has strong affinity to UDP-GalNAc and very low affinity to 2''-fucosyllactose, which is a soluble analog of the H-substances. The low degree of glycosylation of the blood group H-sites due to the low affinity of Aint-enzyme with the H-substances can account for the lower A activity and higher H activity in Aint red cells than in A1 red cells. The blood group A allele can be subdivided into three common alleles, A1, A2, and Aint, each controlling the formation of different types of blood group GalNAc transferases.     

Change of concanavalin A induced agglutinability during preimplantation mouse development

Mouse embryos during early cleavage (zygote to eight-cell stage) were agglutinable with a low concentration (10 μg/ml) of concanavalin A (ConA). This agglutinability was reduced during the first mitotic division. Morulae were agglutinable with a slightly higher concentration (100 μg/ml), whereas blastocysts were not agglutinable even with ConA at a concentration of 5000 μg/ml; however, isolated inner cell masses agglutinated readily at 10 μg/ml of ConA. Embryos grown in vitro behaved as did those isolated directly from the genital tract. Treatment with proteolytic enzymes did not induce agglutinability of mouse blastocyst. The change in agglutinability of trophoblastic cells reflects dramatic changes in the cell surface.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and non-agglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. this was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination.Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg²⁺, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca²⁺ (0.2 mM) had little effect on agglutinability, although high Ca²⁺ (5 mM) inhibited agglutinability of the resealed membranes somewhat.Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells.The agglutination of erythrocytes was not affected by cytochalasin B (40 μg/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes.It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca²⁺ or Mg²⁺ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitive to vinblastine.     

Agglutination of the red blood cells after experimental glycolytic alteration]

The present findings proved Alcian Blue as hemagglutinin suitable for the specific demonstration of negatively charged sites of the cell surface. Erythrocytes with reduced surface charge because of previous trypsination or desialization exhibited decreased Alcian Blue agglutination. On the other hand, Alcian Blue agglutinates more intensely red blood cells with a high surface charge (reticulocytes). Findings with A antiserum or the lectins prepared from Lens culinaris and Helix pomatia indicate a masking of glycolipidic group A receptors by glycoproteins of the erythrocyte membrane. Effects of enzymatic degradation (neuraminidase, trpysin, pronase) combined with incubation in NaF-medium for the deprivation of ATP could be explained by a rearrangement of receptors resulting in altered agglutinability of erythrocytes.     

Surface modification of guinea pig sperm during in vitro capacitation: an assessment using lectin-induced agglutination of living sperm

Plant lectins have been used to advantage to study carbohydrate-containing cell surface receptors in numerous systems. In this study, a simple, reliable assay was developed to quantitate lectin-induced agglutinability of sperm. This assay was used successfully to compare some of the surface properties of uncapacitated and capacitated guinea pig sperm. Capacitation was induced by incubating sperm in minimum capacitation medium (MCM) or modified Tyrodes solution (T-PL). Control incubations were done in Ham's F-10 or Hank's balanced salt solution which do not support capacitation. At timed intervals during incubation, sperm samples were assessed for pattern and degree of lectin-induced agglutination. Results establish that: (1) soybean agglutinin (SBA) and to a lesser extent concanavalin A (Con A) induced agglutinability of guinea pig sperm increase during in vitro capacitation in MCM; (2) a similar increase in SBA induced agglutinability occurs during capacitation in T-PL, but not in the non-capacitating media; and (3) for sperm incubated in MCM or T-PL, there is a significant increase in tail to tail agglutination after capacitation. The results with SBA demonstrate that D-galactose and/or N-acetyl-D-galactosamine containing receptor sites or the guinea pig sperm surface are affected by capacitation, and this effect occurs, at least in part, in the sperm tail. Possible explanations for the observed increase in agglutinability are discussed. The agglutination assay may prove useful as a direct test for the occurrence of capacitation and may be especially valuable for species having a small acrosome or limited number of eggs.