Plasmodium falciparum Cyclic GMP-Dependent Protein Kinase Interacts with a Subunit of the Parasite Proteasome.

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite's cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG's mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG's phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

Invasion of hepatocytes by Plasmodium sporozoites requires cGMP-dependent protein kinase and calcium dependent protein kinase 4.

Invasion of hepatocytes by sporozoites is essential for Plasmodium to initiate infection of the mammalian host. The parasite's subsequent intracellular differentiation in the liver is the first developmental step of its mammalian cycle. Despite their biological significance, surprisingly little is known of the signalling pathways required for sporozoite invasion. We report that sporozoite invasion of hepatocytes requires signalling through two second-messengers - cGMP mediated by the parasite's cGMP-dependent protein kinase (PKG), and Ca2+ , mediated by the parasite's calcium-dependent protein kinase 4 (CDPK4). Sporozoites expressing a mutated form of Plasmodium berghei PKG or carrying a deletion of the CDPK4 gene are defective in invasion of hepatocytes. Using specific and potent inhibitors of Plasmodium PKG and CDPK4, we demonstrate that PKG and CDPK4 are required for sporozoite motility, and that PKG regulates the secretion of TRAP, an adhesin that is essential for motility. Chemical inhibition of PKG decreases parasite egress from hepatocytes by inhibiting either the formation or release of merosomes. In contrast, genetic inhibition of CDPK4 does not significantly decrease the number of merosomes. By revealing the requirement for PKG and CDPK4 in Plasmodium sporozoite invasion, our work enables a better understanding of kinase pathways that act in different Plasmodium stages.

Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development.

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning.

Intraabdominal adhesion formation after preperitoneal dissection in the murine model.

BACKGROUND: The laparoscopic approach to hernia repair has been advocated by many as a potentially superior method of herniorraphy. Several techniques have been described, each with its own proposed advantages. These techniques involve different anatomic approaches, the most recent of which is the totally extraperitoneal approach (TEPA). One presumed advantage of the extraperitoneal approach is the avoidance of adhesion formation because the peritoneum is not entered and mesh is not placed in direct contact with intra-abdominal structures. We hypothesize, however, that when the peritoneum is dissected from the abdominal wall, it is partially devascularized, leading to scar formation and potential adhesion formation. This would suggest that the TEPA method of herniorraphy may not completely avoid the risks of intra-abdominal adhesion formation. METHODS: After appropriate approval was obtained, 88 male Sprague-Dawley rats were divided into two equal groups. One group underwent laparotomy followed by careful blunt dissection of the peritoneum from the left abdominal wall. The control group underwent laparotomy without manipulation of the peritoneum. All animals were re-explored 14 days later, and the abdominal cavity was examined for adhesions. The type and location of any adhesion was recorded. RESULTS: Adhesion formation occurred in 10 of 44 (23%) subjects in the peritoneal dissection group, compared with 3 of 44 (7%) in the nondissection group (p < 0.05). CONCLUSIONS: Dissection of the peritoneum from the overlying abdominal wall in the murine model leads to intra-abdominal adhesion formation. This suggests that peritoneal dissection in the TEPA method of herniorraphy may lead to intra-abdominal adhesion formation.

The Drosophila dCREB-A gene is required for dorsal/ventral patterning of the larval cuticle.

We report on the characterization of the first loss-of-function mutation in a Drosophila CREB gene, dCREB-A. In the epidermis, dCREB-A is required for patterning cuticular structures on both dorsal and ventral surfaces since dCREB-A mutant larvae have only lateral structures around the entire circumference of each segment. Based on results from epistasis tests with known dorsal/ventral patterning genes, we propose that dCREB-A encodes a transcription factor that functions near the end of both the DPP- and SPI-signaling cascades to translate the corresponding extracellular signals into changes in gene expression. The lateralizing phenotype of dCREB-A mutants reveals a much broader function for CREB proteins than previously thought.

A new member of the frizzled family from Drosophila functions as a Wingless receptor.

Receptors for Wingless and other signalling molecules of the Wnt gene family have yet to be identified. We show here that cultured Drosophila cells transfected with a novel member of the frizzled gene family in Drosophila, Dfz2, respond to added Wingless protein by elevating the level of the Armadillo protein. Moreover, Wingless binds to Drosophila or human cells expressing Dfz2. These data demonstrate that Dfz2 functions as a Wingless receptor, and they imply, in general, that Frizzled proteins are receptors for the Wnt signalling molecules.

Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site that involves His-15, which is phosphorylated to form a N delta 1-P-histidine, Arg-17, and the carboxy-terminal residue Glu-85. Mutant HPrs with alterations to the three C-terminal residues, Glu-85, Leu-84, and Glu-83, were produced by site-directed mutagenesis. The properties of these mutants were assessed by kinetic analysis of enzyme I, enzyme IImannose, enzyme IIN-acetylglucosamine, and enzyme IImannitol, and the phosphohydrolysis properties of the HPr mutants. The results show that it is the C-terminal alpha-carboxyl of Glu-85 that is involved in the active site, and this involvement may be restricted to the phosphoryl donor action of HPr. The contribution of this alpha-carboxyl group is modest as the deletion of Glu-85 resulted in the reduction of the enzyme II activity (kcat/Km) to about 33%. Removal of both Glu-85 and Leu-84 yields an HPr that is an impaired substrate of both the enzyme I and enzyme II reactions. Glu-83 appears to have no role in the active site.

Studies on the mechanism of action of the alkaline phosphatase from Dictyostelium discoideum.

The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activity in an uncompetitive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced at alkaline pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a conformational change that renders the enzyme susceptible to irreversible inhibition by phosphate.

The membrane-bound alkaline phosphatase and 5'-nucleotidase activities of vegetative cells of Dictyostelium discoideum.

Earlier reports suggested that the adenosine monophosphate (AMP)- and the p-nitrophenyl phosphate (pNPP)-hydrolyzing activities of Dictyostelium discoideum membrane preparations are due to different proteins. These results have been apparently contradicted by the recent purification to homogeneity of the two activities from culmination phase cells as a single protein [D. R. Armant and C. L. Rutherford (1981) J. Biol. Chem. 256, 12710-12718]. Results presented here from studies on the activities of vegetative cells support the concept of a single protein. Nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Triton X-100 extracts of cell membrane preparations of D. discoideum showed identical migration of pNPPase and AMPase activities. Furthermore, the previously reported different pH optima of the two activities was due to the fact that pH optima are dependent upon the substrate concentration, and the selective solubilization of AMPase from membrane preparations by phospholipase C can probably be accounted for by the finding that phospholipase C preparations from the same commercial source contain 5'-nucleotidase activity. Moreover, there are alterations in the Km and the stability of both AMPase and pNPPase in a strain with a mutationally altered alkaline phosphatase, further supporting the concept that the two activities are due to a single protein. Both substrates serve as transphosphorylation donors demonstrating that the enzyme activity is mechanistically an alkaline phosphatase.

Rhabdomyosarcoma and benign teratoma.

A case of mediastinal embryonal rhabdomyosarcoma occurring with a benign teratoma is reported. In all histologic sections of tumor, the two elements were distinctly separate. This is felt to represent two independently developing tumors that ultimately fused to form a "collision tumor" rather than a sarcoma arising in a benign teratoma.

Effect of 3-O-methyl-D-glucose on the production of glycosidases by Cryptococcus laurentii and Saccharomyces cerevisiae.

3-O-Methyl-D-glucose (3-O-MeGlc) or a mixture of 3-O-MeGlc and glucose stimulate invertase, beta-glucosidase, alpha-glucosidase, and alpha-galactosidase production by Cryptococcus laurentii. They also increase invertase and alpha-glucosidase production by Saccharomyces cerevisiae. The stimulatory effect of 3-O-MeGlc is not caused by competition with glucose for transport nor by a direct action on glycosidases. It is proposed that 3-O-MeGlc acts as a structural rather than as a functional analogue of glucose displacing it from regulatory sites to relieve catabolite repression. Evidence is presented suggesting that intracellular cAMP levels may be related to the effect of 3-O-MeGlc.

Catabolism of 3-O-methyl-D-glucopyranose by Cryptococcus laurentii.

When Cryptococcus laurentii is grown on 3-O-methyl-D-glucopyranose (3-O-MeGlc), two metabolic products are methanol and an extracellular mannan. Hydrolysis of the mannan produced during growth on 3-O-MeGlc labelled with 14C in the 1 or 6 position yields mannose with retention of the labelling pattern in the carbon chain. O-Methyl-labelled substrate yields radioactive methanol. Three possible mechanisms of O-demethylation are ether cleavage, peroxidatic attack, and beta-elimination. The latter mechanism was shown to be the most likely through the use of double-labelled (3H and 14C) 3-O-MeGlc.

Primary malignant lymphoma cutis in an infant: case report and review of literature.

A 6-month-old infant with primary malignant cutaneous lymphoma is presented. The histologic differentiation between benign cutaneous lymphoid hyperplasia and cutaneous malignant lymphoma is discussed. Intensive therapy is warranted at the onset because of early dissemination and poor response to treatment after dissemination.