Antigenic variation is associated with DNA rearrangements in a relapsing fever Borrelia

Borrelia hermsii, an agent of relapsing fever, undergoes antigenic variation in its host. Surface-exposed proteins with differing primary structures determine the serotype of each organism. Using amino acid sequence data from two of these variable proteins, we synthesized two mixed-sequence oligonucleotides and then used the oligonucleotides to probe mRNA and DNA of three isogenic serotypes of B. hermsii. In Northern blots the probes were specific for the mRNA of the homologous serotype. Southern blots revealed two classes of hybridizing fragments: those common to the three serotypes and those specific for a particular serotype. A serotype-specific DNA fragment, which had hybridized to both oligonucleotide probes, was cloned. Subsequent use of the cloned fragment as a probe provided further evidence that antigenic variation in B. hermsii is associated with DNA rearrangements and with occurrence of expression-linked copies of all, or part, of an antigen-specifying gene.     

On the computation of the tertiary structure of globular proteins—IV. Use of secondary structure information

The distance geometry approach for computing the tertiary structure of globular proteins emphasized in this series of papers (Goelet al., J. theor. Biol. 99, 705–757, 1982) is developed further. This development includes incorporation of some secondary structure information—the location of alpha helices in the primary sequence—in the algorithm to compute the tertiary structure of alpha helical globular proteins. An algorithm is developed which estimates the interresidue distances between chain-proximate helices. These distances, in conjunction with the global statistical average distances obtainable from a database of real proteins and determined by the primary sequence of the protein under study, are used to determine the tertiary structure. Five proteins, parvalbumin, hemerythrin, human hemoglobin, lamprey hemoglobin, and sperm whale myoglobin, are investigated. The root mean square (RMS) errors between the calculated structures and those determined by X-ray diffraction range from 4.78 to 7.56 Å. These RMSs are 0.21–2.76 Å lower than those estimated without the secondary structure information. Contact maps and three-dimensional backbone representations also show considerable improvements with the introduction of secondary structure information.     

Compartmentalization of Ca2+ in sickle cells

Control (AA) and sickle cell anemia (SS) erythrocytes were loaded with Ca-chelator (Quin2 or Benz2) to increase the cellular exchangeable Ca2+ pool and to measure the Ca2+ exchange fluxes and the cytosolic ionized Ca2+ ([Ca]i) (Lew et al., 1982, Nature, 298, 478). The chelator incorporation induced a decrease in the ATP content which was smaller in SS than in AA cells and partially reversible upon reincubation in a chelator-free medium. The amount of trapped chelator was determined by two methods: 45Ca binding to the chelator in Ca-ionophore treated cells in Ca-EGTA buffers and [3H]Quin2 incorporation. A slight over-estimation of the chelator content was found with the second method but incorporation was the same in both types of cells. The kinetics of 45Ca equilibration and 45Ca release were used to measure Ca2+ fluxes and [Ca]i in oxygenated chelator-loaded cells. SS cells, as compared to AA cells, exhibited a moderate increase in Ca2+ fluxes (30-75%) but [Ca]i remained in the same range (about 20 nM). Thus the excess of Ca2+ found in SS cells is not available for the Ca2+ pump or the K+ channel a conclusion in agreement with that of Bookchin et al. (1984, Cell Calcium, 5, 277). Analysis of the 45Ca kinetics showed that in AA cells, exchangeable Ca2+ behaved as one compartment. In SS cells, the existence of a second slowly-exchangeable Ca2+ compartment was demonstrated. This latter (3-5 mumol/l cells) was independent of the concentration of the chelator and thus could represent exchangeable Ca2+ enclosed within the intracellular inside-out vesicles recently observed in SS cells (Williamson et al., 1984, J. Cell. Biol., 99, 430a). Alternatively, these two kinetic pools could reflect heterogeneity of the SS cell population.     

Open-field behaviour in chickens: A replication revisited

In an attempt to show that the open field can still be used as a valid measure of fear, Jones (1983) has reported a failure to replicate some of our findings. The present studies show that this was due to procedural and methodological differences. For instance, we found that birds tested in a novel environment behaved quite differently from those, as in Jones' case, which were placed in one resembling the home cage. Moreover, birds housed in isolation for two days prior to testing reacted differently than those, as again in Jones' case, which were reared in isolation from hatching to the time of testing. The results were interpreted as being consistent with our view that open-field behaviour reflects a conflict between the need to reinstate contact with conspecifics on the one hand, and evade predation on the other.     

The postnatal demasculinization of sexual behavior in the Japanese quail (Coturnix coturnix japonica)

Three experiments were performed to analyze the time course of demasculinization in the Japanese quail and to test the activating and organizing effects of estradiol (E2) in adult sexually active birds. In Experiment 1, males and females were castrated at the age of 1 day or 1, 2, 4, and 6 weeks and treated as adults with testosterone (T). The age of castration had no effect on behavior and morphology in males. Plasma gonadotrophins (LH and FSH) were, however, higher in males castrated at or before than in those castrated after 2 weeks of age. This suggests that postnatal testicular secretions have organizing effects on the pituitary activity. Females which were castrated before 1 week of age were less sensitive to the activating effects of T than males, but were not fully demasculinized. The demasculinization of different reproductive characteristics such as male sexual behavior, cloacal gland size, and weight of the syringeal muscles is achieved in females at different times posthatching. In Experiment 2, castration of male and female quail at the ages of 4 days or 4 weeks confirmed that postnatal ovarian secretions contribute to the full behavioral and morphological demasculinization of females. It is easier to elicit mounting in T-treated females when they are tested in their home cage instead of a test arena. This difference was not observed in males. During Experiment 3, it was impossible to demasculinize sexually active adult males or females by treatment with Silastic implants of E2. E2 did not maintain sexual behavior in ovariectomized females showing male sexual behavior when treated with T but maintained the behavior in males.     

Lymphocyte function in experimental African trypanosomiasis. VII. Loss of antigen-nonspecific suppressor-T-cell activity

The extent of immunosuppression occurring in mice infected with the pathogenic African trypanosomes was studied. Spleen cells from Trypanosoma rhodesiense-infected C57BL/6J mice were tested for antigen-nonspecific suppressor-T-cell (Ts) activity after concanavalin A (Con A) treatment in vitro. After exposure to Con A, control and infected mouse spleen cells were added to responder spleen cell cultures stimulated with sheep erythrocytes (SRBC). Assays for the resultant plaque-forming cell responses to SRBC revealed that antigen-nonspecific Ts activity was lost during the first week of infection. Changes in infected mouse T-cell subpopulations, including a terminal loss of Lyt 2.2+ cells, accompanied but did not precede the demonstrable loss of Ts function. Splenic suppressor macrophages which arise during infections with T. rhodesiense also did not seem to be associated with the loss of antigen-nonspecific Ts activity. It is concluded that the generalized immunosuppression associated with experimental African trypanosomiasis extends to the mitogen-induced Ts population.     

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG₁ antibody response was noted after the administration of Con A whereas profound enhancement of IgG_2a antibody response was noted after PHA was given.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.