The Plasmodium translocon of exported proteins component EXP2 is critical for establishing a patent malaria infection in mice

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium translocon of exported proteins (PTEX) and a cleavable Plasmodium export element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic Plasmodium berghei parasites deficient in EXP2, the putative pore‐forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood‐stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL‐bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL‐bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection.     

Opposing effects of PKCalpha and PKCepsilon on basolateral membrane dynamics in intestinal epithelia

PKC is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC are poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKCepsilon followed by late activation of the conventional isoform PKCalpha in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKCepsilon was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKCalpha was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKCalpha on basolateral endocytosis. Inhibition of PKC by the selective conventional PKC inhibitor G?-6976 (5 microM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKCepsilon-selective agonist carbachol (100 microM) induced prolonged stimulation of endocytosis devoid of an inhibitory phase. Actin disassembly caused by PMA was completely blocked by G?-6850 but not by G?-6976, implicating PKCepsilon as the key isoform responsible for actin disruption. The Ca2+ agonist thapsigargin (5 microM) induced early activation of PKC when added simultaneously with PMA. This early activation of PKCalpha blocked the ability of PMA to remodel basolateral F-actin and abolished the stimulatory phase of basolateral endocytosis. Activation of PKCalpha stabilizes F-actin and thereby opposes the effect of PKCepsilon on membrane remodeling in T84 cells.     

Caveolin mediates rapid glucocorticoid effects and couples glucocorticoid action to the antiproliferative program

Many glucocorticoid (Gc) actions are of rapid onset and therefore require acute regulation of intracellular signaling cascades. Integration of diverse extracellular signals requires cross-talk between intracellular pathways, suggesting the existence of nodes for signal interaction, such as the specialized membrane microdomains caveolae. We have identified rapid Gc-dependent phosphorylation of caveolin, and protein kinase B (PKB)/Akt, in the lung epithelial cell line A549 and found this was dependent on src kinases. There was also activation of PKB downstream molecules glycogen synthase kinase-3beta, and mammalian target of rapamycin. Subcellular fractionation colocalized glucocorticoid receptor (GR) and c-src to caveolin-containing membrane fractions. Coimmunoprecipitation studies also identified interactions between GR and caveolin and suggested that the activation function 1 domain within the GR may serve to support an interaction between GR and caveolin. Disruption of lipid raft formation, impairment of caveolin function using dominant-negative caveolin, down-regulation of caveolin-1 using short hairpin RNA or complete ablation of caveolin-1 prevented Gc-induced activation of PKB. Loss of caveolin-1 also prevents Gc activation of glycogen synthase kinase-3beta and mammalian target of rapamycin. In contrast, caveolin interference/down-regulation had no effect on Gc transactivation. Functional analysis of caveolin-1 knockdown and knockout cells identified profound loss of Gc-mediated growth inhibition compared with controls, with a requirement for caveolin in order for Gc to regulate cell cycle progression. Therefore, disruption of caveolae leads to dissociation of Gc action, with impaired induction of PKB activation, and cell growth inhibition, but with negligible effects on Gc transactivation. These observations have implications for understanding the diverse physiological actions of Gc.     

An audit of 'equivocal' (C3) and 'suspicious' (C4) categories in fine needle aspiration cytology of the breast

An audit of 'equivocal' (C3) and 'suspicious' (C4) categories in fine needle aspiration cytology of the breast
We have audited the frequency of use and outcome of the 'equivocal/atypia probably benign' (C3) and 'suspicious of malignancy' (C4) category for breast cytology in our Unit. A total of 14 935 cytological specimens were reported by at least one of the three pathologists with a special interest in breast pathology, according to five categories of the NHSBSP guidelines for cytology reporting, 1992; 3.7% (555 cases) and 3.9% (587 cases) of cases were classified as equivocal (C3) and suspicious (C4), respectively, giving a total rate (C3 + C4) of 7.6%. Of the C3 cases, 68% were subsequently benign and 32% were malignant. Of the C4 cases, 19% were subsequently benign and 81% malignant. The commonest benign lesions in both categories were fibroadenomas (7.6% of C3 and 19.8% of C4), fibrocystic change (14.3% of C3 and 12.5% of C4), radial scars (6.2% of C3 and 10.4% of C4) and papillomas (6.2% of C3 and 6.3% of C4). Of the malignant lesions (particularly those classified as C3), a high proportion were low grade or special type cancers. The categories of atypia probably benign (C3) and suspicious of malignancy (C4) in breast cytology provide a strategy for classification of problematic or uncertain cases; this maintains the predictive value of the benign (C2) and malignant (C5) categories, and allows separation of these difficult cases into clinically useful groups with differing probabilities of malignancy.     

Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim

Refined crystal structures are reported for complexes of Escherichia coli and chicken dihydrofolate reductase containing the antibiotic trimethoprim (TMP). Structural comparison of these two complexes reveals major geometrical differences in TMP binding that may be important in understanding the stereo-chemical basis of this inhibitor's selectivity for bacterial dihydrofolate reductases. For TMP bound to chicken dihydrofolate reductase we observe an altered binding geometry in which the 2,4-diaminopyrimidine occupies a position in closer proximity (by approximately 1 A) to helix alpha B compared to the pyrimidine position for TMP or methotrexate bound to E. coli dihydrofolate reductase. One important consequence of this deeper insertion of the pyrimidine into the active site of chicken dihydrofolate reductase is the loss of a potential hydrogen bond that would otherwise form between the carbonyl oxygen of Val-115 and the inhibitor's 4-amino group. In addition, for TMP bound to E. coli dihydrofolate reductase, the inhibitor's benzyl side chain is positioned low in the active-site pocket pointing down toward the nicotinamide-binding site, whereas, in chicken dihydrofolate reductase, the benzyl group is accommodated in a side channel running upward and away from the cofactor. As a result, the torsion angles about the C5-C7 and C7-C1' bonds for TMP bound to the bacterial reductase (177 degrees, 76 degrees) differ significantly from the corresponding angles for TMP bound to chicken dihydrofolate reductase (-85 degrees, 102 degrees). Finally, when TMP binds to the chicken holoenzyme, the Tyr-31 side chain undergoes a large conformational change (average movement is 5.4 A for all atoms beyond C beta), rotating down into a new position where it hydrogen bonds via an intervening water molecule to the backbone carbonyl oxygen of Trp-24.     

Effects of distal point-site mutations on the binding and catalysis of dihydrofolate reductase from Escherichia coli

The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis. The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C. Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected. Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively). Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L. The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 A away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to greater than 45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 microM for H45Q (similar to wild type) but from 8 to 5000 microM for R44L.     

Synergism in folding of a double mutant of the alpha subunit of tryptophan synthase

The urea-induced unfolding of the inactive single mutants Tyr-175----Cys and Gly-211----Glu and the active double mutant Cys-175/Glu-211 of the alpha subunit of tryptophan synthase from Escherichia coli was examined by using ultraviolet difference spectroscopy. Equilibrium techniques were used to determine the equilibrium free energies of unfolding for the mutant proteins to permit comparison with the wild-type protein. The sum of the changes in stability for the single mutants is not equal to the change seen in the double mutant. This inequality is evidence for a structural interaction between these two residues. Kinetic studies show that this synergism, which destabilizes the native form by 1.5-2.0 kcal/mol at pH 7.8, 25 degrees C, occurs only after the final rate-limiting step of domain association.     

Microsecond hydrophobic collapse in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein

Using small-angle X-ray scattering combined with a continuous-flow mixing device, we monitored the microsecond compaction dynamics in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein. A significant collapse of the radius of gyration from 30 A to 23.2 A occurs within 300 micros after the initiation of refolding by a urea dilution jump. The subsequent folding after the major chain collapse occurs on a considerably longer time-scale. The protein folding trajectories constructed by comparing the development of the compactness and the secondary structure suggest that the specific hydrophobic collapse model rather than the framework model better explains the experimental observations. The folding trajectory of this alpha/beta-type protein is located between those of alpha-helical and beta-sheet proteins, suggesting that native structure determines the folding landscape.     

The temperatures of nine flowing waters in southern England

The authors analyse water temperatures in seven streams and two watercress beds in southern England, and examine some of the biological implications.     

Immunisation of cattle with recombinant acetylcholinesterase from Dictyocaulus viviparus and with adult worm ES products

Dictyocaulus viviparus causes a serious lung disease of cattle. For over 30 years, a radiation-attenuated larval vaccine has been used with success; however, this vaccine has several disadvantages. A more stable vaccine against D. viviparus, capable of stimulating prolonged protective immunity, would be beneficial. Recent research has been directed at adult worm ES components that may be involved in parasite survival in the host. One component is the secreted enzyme, acetylcholinesterase (AChE), a target for circulating antibody in infected calves. Here, we describe a study where protection was investigated in calves immunised with either native adult ES products or a recombinant parasite AChE. These antigens were administered twice with Freund's incomplete adjuvant. Subsequently, all calves were challenged with 700 L3 and their worm burdens and immune responses compared with those in calves that received an anthelmintic-abbreviated infection and challenge control calves. Significant levels of protection were not obtained in the immunised groups but significant immunity was achieved in the calves that received the anthelmintic abbreviated infection. Antibody responses amongst the groups were different, with significantly higher IgG1 responses in the immune, infected group and in adult ES recipients. Significantly higher IgG2 responses were found in the latter group. Following challenge, the groups that received the abbreviated infection and the fusion protein produced specific antibody that bound the native enzyme. No differences were observed between groups in peripheral blood mononuclear cell responsiveness to either antigen. However, adult ES products appeared to have a mitogenic effect on these cells, whilst the fusion protein exhibited an inhibitory effect. These results suggest that in this form, AChE is not a potential vaccine candidate and that adult ES products, in contrast to previous experiments in guinea pigs, do not contain protective components.