Expression of the cytolethal distending toxin (Cdt) operon in Actinobacillus actinomycetemcomitans: evidence that the CdtB protein is responsible for G2 arrest of the cell cycle in human T cells

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several gram-negative bacteria. In this study, we report that the cdt locus in A. actinomycetemcomitans is composed of five open reading frames, designated orf1, orf2, cdtA, cdtB, and cdtC. The deduced amino acid sequences of the five open reading frames are highly conserved among A. actinomycetemcomitans strains 652, Y4, 29522, and HK1651. There is also strong homology with the Cdt proteins of Haemophilus ducreyi (87-91%), but only partial homology with that of Campylobacter jejuni and Escherichia coli (29-48%). Analysis of A. actinomycetemcomitans mRNA by RT-PCR suggests that the two small open reading frames upstream of cdtA are coexpressed with cdtA, cdtB, and cdtC. We next utilized a series of plasmids that express various combinations of the cdt genes to determine their requirement for expression of immunoinhibitory activity. Cell extracts of E. coli transformed with each of the plasmids were tested for their capacity to induce G2 arrest in the cell cycle of PHA-activated human T cells. These experiments suggest that expression of cdtB alone is sufficient to induce G2 arrest in human T cells, but do not exclude the possibility that cdtC also contributes to cell cycle arrest. The implications of our results with respect to the function of the individual Cdt proteins are discussed.     

Inhibition of antigen-induced eosinophilia and late phase airway hyperresponsiveness by an IL-5 antisense oligonucleotide in mouse models of asthma

Chronic airway eosinophilia is associated with allergic asthma and is mediated in part by secretion of IL-5 from allergen-specific Th2 lymphocytes. IL-5 is a known maturation and antiapoptotic factor for eosinophils and stimulates release of nascent eosinophils from bone marrow into the peripheral circulation. An antisense oligonucleotide found to specifically inhibit IL-5 expression in vitro was observed to significantly reduce experimentally induced eosinophilia in vivo, in both the murine OVA lung challenge and allergic peritonitis models. Intravenous administration resulted in sequence-dependent inhibition of eosinophilia coincident with reduction of IL-5 protein levels, supporting an antisense mechanism of action. Potent suppression of lung eosinophilia was observed up to 17 days after cessation of oligonucleotide dosing, indicating achievement of prolonged protection with this strategy. Furthermore, sequence-specific, antisense oligonucleotide-mediated inhibition of Ag-mediated late phase airway hyperresponsiveness was also observed. These data underscore the potential utility of an antisense approach targeting IL-5 for the treatment of asthma and eosinophilic diseases.     

Stress renders T cell blasts sensitive to killing by activated syngeneic NK cells

Exposure of primary T cell blasts to stress in the forms of heat, hydrogen peroxide, or high-density growth conditions resulted in a state of enhanced susceptibility to killing by syngeneic IL-2-activated NK cells or lymphokine-activated killer cells, but not to killing by CTL. Cytotoxicity was perforin mediated and was not due to decreased target expression of total MHC class I. The levels of stress used had little effect on cell viability. For thermal stress, sensitization increased with temperature, required a minimum exposure time, and disappeared when cells were given a long enough recovery time. Our data support a model that predicts that activated NK cells play a role in the immunosurveillance of nontransformed stressed cells in normal animals.     

Production of a recombinant form of early pregnancy factor that can prolong allogeneic skin graft survival time in rats

Early pregnancy factor (EPF), an extracellular chaperonin 10 homologue, has immunosuppressive and growth factor properties. In order to carry out more extensive studies on the in vivo characteristics of EPF, a recombinant form of the molecule has been prepared. Recombinant human EPF (rEPF) was expressed in Escherichia coli using the plasmid pGEX-2T expression system. Potency of rEPF in vitro in the rosette inhibition test, the bioassay for EPF, was equivalent to that of native EPF (nEPF), purified from human platelets, and synthetic EPF (sEPF). However, the half-life of activity (50% decrease in the log value) in serum, following i.p. injection, was significantly decreased (3.2 h, compared with nEPF 6.2 days, sEPF 5.8 days). This was thought to be due to modification of the N-terminus of the recombinant molecule inhibiting binding to serum carrier proteins. Because EPF can modify Th1 responses, the ability of the recombinant molecule to suppress allogeneic graft rejection was investigated. Following skin grafts from Lewis rats to DA rats and vice versa, rEPF was delivered locally at the graft site and the effect on survival time of the allografts noted. Results demonstrated that rEPF treatment significantly prolonged skin graft survival time by as much as 55% in stringent models of transplantation across major histocompatibility barriers.     

STAT3 regulates the growth and immunoglobulin production of BCL(1) B cell lymphoma through control of cell cycle progression

STAT3 is constitutively phosphorylated on tyrosine(705) in self-renewing, CD5(+) murine B-1 lymphocytes. Nuclear extracts from untreated primary B-1 or CD5(+) BCL(1) B lymphoma cells were found to contain immunoreactive STAT3 protein that binds to a sis-inducible element present in the promoter of the p21(waf1/cip1) tumor suppressor gene and is constitutively phosphorylated on serine(727). To determine the functional significance of constitutive STAT3 activation in B lymphoma cells, a specific STAT3 antisense oligonucleotide was developed and used to examine basal BCL(1) cell growth and IgM production. Abrogating STAT3 expression in BCL(1) cells inhibited their proliferative capacity and induced a corresponding decrease in secretion of IgM. Cell cycle analysis showed a block in progression through G1 in BCL(1) cells treated with the STAT3 antisense oligonucleotide. These results indicate that STAT3 controls cell growth and immunoglobulin secretion by enhancing progression through the G1 phase of the cell cycle in BCL(1) B cell lymphoma.     

Sol-gel trapping of functional intermediates of hemoglobin: geminate and bimolecular recombination studies

The encapsulation of proteins in porous sol-gels is a promising technique for generating, trapping, and probing functionally significant nonequilibrium protein species. An essential step needed in the pursuit of that goal is establishing the degree to which the sol-gel limits conformational change upon adding or removing substrates. In the present study, geminate recombination and solvent phase bimolecular recombination of CO to human adult hemoglobin (HbA) are used as sensitive probes of the degree of conformational constraint within the sol-gel. Two forms of CO saturated encapsulated HbA are generated. In one case, designated [COHbA], the equilibrium form of COHbA is directly encapsulated. In the second case, designated as [deoxyHbA] + CO, the equilibrium form of deoxyHbA is encapsulated and only after the sample has aged is CO introduced to the HbA through the porous sol-gel matrix. Three different preparative protocols are used to generate the sol-gels for each of the two forms of encapsulated COHbA. The kinetic traces obtained from these encapsulated samples allow for an easy evaluation of the extent to which the sol-gel is locking in the initial tertiary/quaternary structure. The results show that the sol-gel encapsulated samples can be used with pulsed laser sources and that one of the tested encapsulation protocols is far superior with respect to conformational locking. This protocol is used to trap and probe nonequilibrium forms such as the liganded T state of HbA, a species whose properties are needed to fully explore allostery in HbA.     

Quantitative trait loci affecting differences in floral morphology between two species of monkeyflower (Mimulus).

Conspicuous differences in floral morphology are partly responsible for reproductive isolation between two sympatric species of monkeyflower because of their effect on visitation of the flowers by different pollinators. Mimulus lewisii flowers are visited primarily by bumblebees, whereas M. cardinalis flowers are visited mostly by hummingbirds. The genetic control of 12 morphological differences between the flowers of M. lewisii and M. cardinalis was explored in a large linkage mapping population of F2 plants n = 465 to provide an accurate estimate of the number and magnitude of effect of quantitative trait loci (QTLs) governing each character. Between one and six QTLs were identified for each trait. Most (9/12) traits appear to be controlled in part by at least one major QTL explaining >/=25% of the total phenotypic variance. This implies that either single genes of individually large effect or linked clusters of genes with a large cumulative effect can play a role in the evolution of reproductive isolation and speciation.     

Passive Ca binding in ventricular myocardium of neonatal and adult rats

In this study, passive Ca²⁺ binding was determined in ventricular homogenates (VH) from neonatal (4–6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca²⁺ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca²⁺ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca²⁺] ([Ca²⁺]_F was measured with Indo-1 and bound Ca²⁺ ([Ca²⁺]_B) was the difference between [Ca²⁺]_F and total Ca²⁺. Apparent Ca²⁺ dissociation constants (K_d) for BAPTA and Indo-1 were increased by 10–20 mg VH protein/ml (from 0.35 to 0.92 μM for Indo-1 and from 0.20 to 0.76 μM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl₂ additions (2.5–10 nmoles) yielded δ[Ca²⁺]_B/δ[Ca²⁺]_F for the sum of [Ca²⁺]_B at all three classes of binding sites. From this function, the apparent number of endogenous sites (B_en) and their K_d (K_en) were determined. Similar K_en values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 ± 0.14 μM, 0.69 ± 0.13 μM and 0.53 ± 0.10 μM, respectively). However, B_en was significantly higher in adult myocytes than in adult VH (1.73 ± 0.35 versus 0.70 ± 0.12 nmol/mg protein, P < 0.01), which correspond to ∼300 and 213 μmol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that B_en determined in VH underestimates cellular B_en by 29%. Although B_en values in nmol/mg protein were similar in adult and neonatal VH (0.69 ± 0.12), protein content was much higher in adult ventricle (125 ± 7 versus 80 ± 1 mg protein/g wet weight, P < 0.01). Expressing B_en per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca²⁺ binding capacity at high-affinity sites is ∼300 and 176 mmol/I cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca²⁺ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month.