Human T cell responses to gp63, a surface antigen of Leishmania

gp63, an abundant and conserved leishmania cell surface protein, has been implicated in the ability of these parasitic protozoa to infect macrophages in vitro and has shown potential as a protective immunogen in mice. However, little is known regarding human immune responses to this glycoprotein Ag. In this study, human T lymphocyte responses to Leishmania amazonensis native gp63 and to recombinant gp63 (rgp63) produced in Escherichia coli were evaluated in individuals with active or cured cutaneous, mucosal or visceral leishmaniasis. Both native and rgp63 elicited strong proliferative responses in all patients tested. In addition, IFN-gamma was produced in response to stimulation with both forms of the protein. T cell lines generated from PBMC by stimulation with native or rgp63 were phenotypically similar, and proliferated and produced IFN-gamma in response to stimulation with both forms of the molecule. These results suggest that gp63 is a strong T cell immunogen and that the recombinant and native forms can elicit the same type of T cell response from infected patients. In order to compare the immunogenic properties of these two forms of gp63, PBMC from naive (uninfected) donors were sensitized in vitro with native or rgp63. T cell lines generated against rgp63 proliferated in response to rgp63, but failed to proliferate in response to native gp63 or to promastigote lysate. Thus, rgp63 was effective in eliciting T cell responses from patients with active or cured leishmania infection, but did not effectively induce T cell responses under the conditions used.     

Predation by a water mite (Piona exigua) on enclosed populations of zooplankton

Short term, replicated experimental alteration of densities of a predatory water mite Piona exigua Viets, inside 0.45–0.475 m³ litre enclosures, revealed little evidence of the effects of predation on the number and relative abundance of the enclosed zooplankton species. Predation rates more closely approximated those estimated from single prey functional response experiments in the second experimental period (December) than in the first (March). In December Daphnia was the only susceptible taxon present in large numbers, whereas in March, Ceriodaphnia and Chydorus were also present. This result is consistent with laboratory findings that predation rates are lowered in the presence of more than one prey type.The difficulty of obtaining evidence for significant effects of these planktonic predators is in part due to changes in the preferred prey species in the diet of Piona depending on stage and sex of the mite and to aspects of experimental design. The wide variability between replicate enclosures at each predator density reduced the power of the statistical analyses used to test the null hypothesis. Enclosures with no predators are necessary to investigate the effects of enclosure on the zooplankton prey, since these effects may outweigh those due to predator consumption.     

Primary structure of kunitz-type trypsin inhibitor-2a (pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed

The primary structure of acidic trypsin inhibitor-2a (WBTI-2a,pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed was determined. This inhibitor consists of a single polypeptide chain of 180 amino acids including four half-cystine residues and has an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2a,pI 5.9, showed 84% identity to acidic trypsin inhibitor-2 (WBTI-2,pI 5.1) but only 57% identity to the basic trypsin inhibitor (WBTI-1,pI 8.9) and 50% identity to the chymotrypsin inhibitor of winged bean. The data indicate that winged bean seed contains a family of three Kunitz-type inhibitors which have about 50% identity.     

Interaction of pertussis toxin with cells and model membranes.

The interaction of pertussis toxin (PT) with cells and model membranes was investigated by examining PT-induced intoxication of Chinese hamster ovary cells and by studying the binding of PT and its subunits to phospholipid vesicles. Since certain bacterial toxins require an acidic environment for efficient interaction with membranes and subsequent entry into the cell, the requirement for an acidic environment for PT action was examined. PT, unlike bacterial toxins such as diphtheria toxin, did not require an acidic environment for efficient intoxication of Chinese hamster ovary cells. Potential modes by which PT might interact with biological membranes were studied by examining the binding of PT to a model membrane system. PT was found to be capable of interacting with phospholipid vesicles, however, efficient binding of the toxin to the vesicles occurred only in the presence of both ATP and reducing agent. The A subunit portion of the toxin bound preferentially to the vesicles while little binding of the B oligomer portion of PT to the model membranes was observed. Isolated A subunit, in the absence of the B oligomer, also bound to the vesicles with optimal binding occurring in the presence of reducing agent. After cleavage of the A subunit by trypsin, probably at Arg-181, Arg-182, and/or Arg-193, large fragments which lacked the C-terminal portion of the A subunit of PT no longer associated with the lipid vesicles. These results suggest that the A subunit of PT can interact directly with a lipid matrix and, if freed from the constraints imposed by the B oligomer, may be capable of interacting with cellular membranes.     

Immunochemical localization of a region of chaperonin-60 important for productive interaction with chaperonin-10.

An IgG1 monoclonal antibody (mAb 54G8) which binds to both Bordetella pertussis chaperonin-60 (cpn60) and Escherichia coli cpn60 (GroEL) was produced. mAb 54G8 as well as Fab fragments prepared from this antibody were found to abolish the ability of chaperonin-10 (cpn10, GroES) to inhibit the ATPase activity of both B. pertussis cpn60 and E. coli cpn60. Electron microscopy was used to localize the binding site of the monoclonal antibody on the B. pertussis cpn60 molecule. In the absence of the antibody, the B. pertussis molecule exhibited the tetradecameric structure typical of cpn60. Both end views (showing 7-fold symmetry of the face of the molecule) and side views were evident. When mAb 54G8 was bound, B. pertussis cpn60 molecules appeared to be cross-linked so that they formed long chains. Only side views of the molecules were seen in these long chains. When B. pertussis cpn60 complexed with Fab fragments of mAb 54G8 was examined, chains were no longer observed. Instead, side views of B. pertussis cpn60 were often seen with Fab fragments extending from the ends of the molecule. These data indicate that mAb 54G8 appears to bind at or near the end of the B. pertussis cpn60 molecule and that binding of mAb 54G8 at this location affects the ability of cpn10 to productively interact with cpn60, most likely either by sterically blocking the binding of cpn10, by affecting the conformation of cpn60 in such a way that it no longer binds cpn10, or by inhibiting proper transduction of the effects of cpn10 binding.     

Influence of plant nutrient concentration on growth rate: Use of a nutrient interruption technique to determine critical concentrations of N,P and K in young plants

A method is described for determining the way in which growth rate varies with plant nutrient concentration using a simple nutrient interruption technique incorporating only 2 treatments. The method involves measuring the changes in growth and nutrient composition of otherwise well-nourished plants after the supply of one particular nutrient has been withheld. Critical concentrations are estimated from the relationship between the growth rate (expressed as a fraction of that for control plants of the same size which remained well-nourished throughout) and the concentration of the growth-limiting nutrient in the plants as deficiency developed. Trials of the method using young lettuce plants showed that shoot growth rate was directly proportional to total N (nitrate plus organic N) concentration, and linearly or near-linearly related to K and P concentration over a wide range; the corresponding relationship for nitrate was strongly curvi-linear. Critical concentrations (corresponding to a 10% reduction in growth rate) determined from these results were similar to critical values calculated from models derived from field data, but were generally higher than published estimates of critical concentration (based on reductions in shoot weight) for plants of a similar size. Reasons for these discrepancies are discussed. Nitrate, phosphate or potassium concentrations in sap from individual leaf petioles were highly sensitive to changes in shoot growth rate as deficiency developed, with the slope of the relationships varying with leaf position, due to differences both in their initial concentration and in the rates at which they were utilized in individual leaves. Each nutrient was always depleted more quickly in younger leaves than in older ones, providing earlier evidence of deficiency for diagnostic purposes. Although the plants were capable of accumulating nitrate, phosphate and potassium well in excess of that needed for optimum dry matter production during periods of adequate supply, the rate of mobilization of these reserves was insufficient to prevent reductions in growth rate as the plants became deficient. This brings into question the validity of the conventional concept that luxury consumption provides a store of nutrients which are freely available for use in times of shortage. The implications of these results for the use of plant analysis for assessing plant nutrient status are discussed.     

Covalent immobilization of laccase on activated carbon for phenolic effluent treatment

Summary Laccase was covalently immobilised to activated carbon using four derivatisation methods. The highest bound activity was obtained using diimide coupling of laccase to carboxyl groups on the carbon. The maximum bound activity was reached at 11.5 mg laccase/g carbon. The carbon-immobilised laccase (CIL) was stable at pH values from 4.0 to 9.0. CIL stored at 4°C lost 38± 5% activity in the first 4 days, then a further 22±5% in 126 days. CIL showed increased stability to low pH although the pH optimum was unchanged. The activation energy of CIL was lower than soluble laccase. Oxidation of 2,6-dimethoxyphenol (DMP) by CIL in a packed-bed system was only 30±10% of that in a fluidised bed system. Of the initial activity 10–30% was retained after oxidation of seven batches of DMP. CIL removed colour from two industrial effluents. Colour was removed from pulp mill bleach plant effluent at 115 colour units per enzyme unit per hour and the removal rate increased with increasing effluent concentration. Correspondence to: R. G. Burns     

Continuous cell suspension processing using magnetically stabilized fluidized beds

The behavior of a cell suspension in a continuous magnetically stabilized fluidized bed (MSFB) was investigated both experimentally and theoretically. The low, constant pressure drop and fluidity of the solids phase in the MSFB allowed a continuous countercurrent separator to be constructed. The magnetic field eliminated all motion of the solids phase (nickel spheres) and produced a device similar to a packed-bed depth filter. Yeast cells were used as the suspended solids and the performance of the MSFB filter was assessed as a function of the bed height, solids velocity, cell concentration, and liquid composition. Removal rates could be adjusted by controlling the cell/support interaction and were found to be as high as 99%. A mathematical model was used to aid in understanding this filtration and was found to agree qualitatively with all experimental observations. Comparison of the model with the data suggests that both cell/cell binding and cell shadowing are occurring.     

Multiple shoot production from seedling explants of slash pine (Pinus elliottii,Engelm.)

Hypocotylary explants obtained from 30- to 40-day-old slash pine (Pinus elliottii, Engelm.) seedlings treated with 6-benzylaminopurine produced multiple buds that eventually elongated into axillary shoots. The explants were pulse treated (45-s dip) with 6-benzylaminopurine (22.2, 111, 222 M) plus a control and cultured on three different basal media containing activated charcoal (0.5% w/v). Hormonal concentration and basal medium were compared for the number and size of axillary shoots induced after 12 and 29 days. The greatest number of axillary shoots was produced by explants that were pulse treated with 111 M 6-benzylaminopurine and cultured on Gresshoff and Doy medium. The axillary shoots were fewer in number per explant than shoots previously reported resulting from hormonally induced advantitious buds of slash pine, but the axillary shoots developed more rapidly.Abbreviations BA 6-benzylaminopurine - DMSO dimethyl sulfoxide - PAR photosynthetically active radiation     

Expression of the alpha, beta II, and gamma protein kinase C isozymes in the baculovirus-insect cell expression system. Purification and characterization of the individual isoforms

Detailed in vitro comparisons of the biochemical characteristics of three protein kinase C isozymes were performed. As an alternative to earlier uncertain separation methods and expression schemes, highly purified and genetically distinct protein kinase C enzymes were produced using the baculovirus expression system. The baculovirus expression system yielded approximately 200-300 micrograms of the purified isozyme from 3 x 10(8) (100 ml of culture medium) baculovirus-infected insect cells. Biochemical characterization of the expressed isozymes indicated that the three isozymes had virtually indistinguishable Ca2+, Mg2+, and ATP dependencies. However, in certain critical functional characteristics such as phosphatidylserine dependencies, phospholipid and substrate preferences, and arachidonic acid activation, the gamma isozyme exhibited distinctive properties when compared with both the alpha and beta II subtypes. In addition, the activity of the beta II subtype was more dependent upon diacylglycerol or phorbol esters for activation than either the alpha or gamma isoforms. The alpha isozyme, unlike the beta II and gamma forms, was totally dependent on Ca2+ for activation in the presence of free arachidonic acid. These studies provide definitive characterizations of the pure isoforms; many of the findings were consistent with earlier enzymatic observations using hydroxyapatite-purified isoforms. Thus, the distinctive biochemical properties of the protein kinase C isozymes are consistent with the hypothesis that each isoform may have distinct roles in signal transduction processes.