A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway

Continuous growth and development in plants are accomplished by meristems, groups of undifferentiated cells that persist as stem cells and initiate organs. While the structures of the apical and floral meristems in dicotyledonous plants have been well described, little is known about the underlying molecular mechanisms controlling cell proliferation and differentiation in these structures. We have shown previously that the CLAVATA1 (CLV1) gene in Arabidopsis encodes a receptor kinase-like protein that controls the size of the apical and floral meristems. Here, we show that KAPP, a gene encoding a kinase-associated protein phosphatase, is expressed in apical and young floral meristems, along with CLV1. Overexpression of KAPP mimics the clv1 mutant phenotype. Furthermore, CLV1 has kinase activity: it phosphorylates both itself and KAPP. Finally, KAPP binds and dephosphorylates CLV1. We present a model where KAPP functions as a negative regulator of the CLAVATA1 signal transduction pathway.     

The Arabidopsis CHL1 protein plays a major role in high-affinity nitrate uptake

The CHL1 (NRT1) gene of Arabidopsis encodes a nitrate-inducible nitrate transporter that is thought to be a component of the low-affinity (mechanism II) nitrate-uptake system in plants. A search was performed to find high-affinity (mechanism I) uptake mutants by using chlorate selections on plants containing Tag1 transposable elements. Chlorate-resistant mutants defective in high-affinity nitrate uptake were identified, and one had a Tag1 insertion in chl1, which was responsible for the phenotype. Further analysis showed that chl1 mutants have reduced high-affinity uptake in induced plants and are missing a saturable component of the constitutive, high-affinity uptake system in addition to reduced low-affinity uptake. The contribution of CHL1 to constitutive high-affinity uptake is higher when plants are grown at more acidic pH, conditions that increase the level of CHL1 mRNA. chl1 mutants show reduced membrane depolarization in root epidermal cells in response to low (250 microM) and high (10 mM) concentrations of nitrate. Low levels of nitrate (100 microM) induce a rapid increase in CHL1 mRNA. These results show that CHL1 is an important component of both the high-affinity and the low-affinity nitrate-uptake systems and indicate that CHL1 may be a dual-affinity nitrate transporter.     

A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.     

A non-specific lipid transfer protein from Arabidopsis is a cell wall protein

Lipid transfer proteins (LTPs), mediate the transfer of phospholipids between membranes in vitro. However, the in vivo function of LTPs is not known. To determine the precise location of a non-specific LTP from Arabidopsis, a cDNA clone was used to produce an Arabidopsis LTP:protein A fusion. Antibodies raised against the fusion were used to localize the Arabidopsis LTP by immunoelectron microscopy. LTP was found to be located in the cell wall, mainly in epidermal cells. This location appears to be inconsistent with the proposed role of the protein in intracellular lipid transfer.     

Pancreatitis-associated protein: a putative marker for pancreas graft rejection

BACKGROUND: Graft rejection is one of the major causes of graft loss after pancreas transplantation. Pancreatitis-associated protein (PAP) is synthesized by the pancreas due to pancreatic inflammation and has shown to be a good serum marker for injury of the pancreas. It may also be potentially useful in the early recognition of rejection and may thus improve pancreas survival. METHODS: We retrospectively evaluated PAP as an early serum marker of pancreas graft rejection in a cross-sectional study in which immunohistochemical analysis of pancreas biopsies was undertaken using antibodies against PAP. PAP concentrations were also measured in sera of blood donors and in patients with renal failure, renal replacement therapy, kidney transplantation alone, and simultaneous pancreas-kidney transplantation. RESULTS: All patients had elevated PAP serum levels compared with blood donors (median PAP: 22 ng/ml, range: 5-75 ng/ml; P<0.0001). Patients on renal replacement therapy had higher values than patients with renal failure (median: 420 ng/ml and 150 ng/ml, respectively). There was a strong inverse correlation between PAP levels and creatinine clearance (P<0.001). PAP values in simultaneous pancreas-kidney transplantation patients with histological rejection were significantly higher than values in those who were clinically stable (median: 925 ng/ml and 322 ng/ml, respectively; P=0.006). Rejection was significantly associated with PAP staining of acinar cell surface. There was also a significant correlation between surface positivity of staining and serum PAP levels (P=0.008). No positive PAP staining was observed in concurrently collected biopsies of renal allografts undergoing rejection. CONCLUSIONS: Serum PAP levels appear to strongly correlate with creatinine clearance measurements. In patients with a pancreas-kidney transplantation, PAP may prove to be a useful biological and histological marker of pancreatic graft rejection.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

A plasma membrane-bound putative endo-1,4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis

Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.     

A putative vacuolar cargo receptor partially colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis roots

Targeting of protein cargo to the vacuole/lysosome is a multistep process that appears to have conserved features between mammalian, yeast, and plant cells. In each case, some soluble vacuolar/lysosomal proteins are believed to be bound by transmembrane cargo receptors in the trans-Golgi network (TGN) that redirect these proteins into clathrin-coated vesicles. These vesicles then appear to be transported to the prevacuole/endosome by a trafficking machinery that requires components identified in other vesicle-targeting steps such as N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment protein (SNAP), SNAP receptors (SNAREs), rab-type GTPases, and Sec1p homologs. Two likely members of this trafficking machinery have been characterized from Arabidopsis thaliana: AtPEP12p, a t-SNARE that resides on a what we now call a prevacuolar compartment, and AtELP, a protein that shares many common features with mammalian and yeast transmembrane cargo receptors. Here, we have further investigated the intracellular distribution of AtELP. We have found that AtELP is located at the trans-Golgi of Arabidopsis root cells, and that its C terminus can preferentially interact in vitro with the mammalian TGN-specific AP-1 clathrin-adapter complex, suggesting a likely role in clathrin-coated, vesicle-directed trafficking at the TGN. Further, consistent with a role in trafficking of vacuolar cargo, we have found that AtELP partially colocalizes with AtPEP12p on a prevacuolar compartment.     

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.     

Cytolytic activity in the genus Leishmania: involvement of a putative pore-forming protein

We describe here that parasites of the genus Leishmania contain a cytolytic activity which acts optimally at pH 5.0 to 5.5 and at 37 degrees C in vitro. or the four species examined, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) major presented considerable hemolytic activity, whereas Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis showed little and no hemolytic activity, respectively. The cytolytic factor of L. amazonensis promastigotes was characterized as a protein with no protease-, phospholipase-, or detergent-like activity, probably localized inside membranous vesicles. The use of osmotic protectants revealed the colloid-osmotic nature of hemolysis, which is indicative of pore formation in the membranes of target cells. This putative pore-forming protein also damaged nucleated cells, including macrophages, causing an increase in their membrane permeability with leakage of cytoplasmic proteins. Both promastigotes and amastigotes express this lytic activity, suggesting that the cytolysin may have a function in both stages of this parasite. The pH and temperature required for optimal activity indicate that it might be more effective within the mammalian host, particularly inside the macrophage parasitophorous vacuole. In promastigotes of L. amazonensis, the expression of lytic activity seems to be regulated during their growth in vitro, being maximal at the early stationary phase.     

A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana

The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, the maintenance of seed dormancy, and the inhibition of plant growth. All three responses are affected in the ABA-insensitive mutant abi1 of Arabidopsis thaliana, suggesting that an early step in the signaling of ABA is controlled by the ABI1 locus. The ABI1 gene was cloned by chromosome walking, and a missense mutation was identified in the structural gene of the abi1 mutant. The ABI1 gene encodes a protein with high similarity to protein serine or threonine phosphatases of type 2C with the novel feature of a putative Ca2+ binding site. Thus, the control of the phosphorylation state of cell signaling components by the ABI1 product could mediate pleiotropic hormone responses.     

Demyelination: the role of reactive oxygen and nitrogen species

This review summarises the role that reactive oxygen and nitrogen species play in demyelination, such as that occurring in the inflammatory demyelinating disorders multiple sclerosis and Guillain-Barré syndrome. The concentrations of reactive oxygen and nitrogen species (e.g. superoxide, nitric oxide and peroxynitrite) can increase dramatically under conditions such as inflammation, and this can overwhelm the inherent antioxidant defences within lesions. Such oxidative and/or nitrative stress can damage the lipids, proteins and nucleic acids of cells and mitochondria, potentially causing cell death. Oligodendrocytes are more sensitive to oxidative and nitrative stress in vitro than are astrocytes and microglia, seemingly due to a diminished capacity for antioxidant defence, and the presence of raised risk factors, including a high iron content. Oxidative and nitrative stress might therefore result in vivo in selective oligodendrocyte death, and thereby demyelination. The reactive species may also damage the myelin sheath, promoting its attack by macrophages. Damage can occur directly by lipid peroxidation, and indirectly by the activation of proteases and phospholipase A2. Evidence for the existence of oxidative and nitrative stress within inflammatory demyelinating lesions includes the presence of both lipid and protein peroxides, and nitrotyrosine (a marker for peroxynitrite formation). The neurological deficit resulting from experimental autoimmune demyelinating disease has generally been reduced by trial therapies intended to diminish the concentration of reactive oxygen species. However, therapies aimed at diminishing reactive nitrogen species have had a more variable outcome, sometimes exacerbating disease.     

Localization of granzyme B in the nucleus. A putative role in the mechanism of cytotoxic lymphocyte-mediated apoptosis

One mechanism used by cytotoxic T cells and natural killer cells to kill target cells involves synergy between the pore-forming protein, perforin, and a serine protease termed granzyme B, both constituents of the cytoplasmic granules of cytolytic lymphocytes. Exposing susceptible cells to perforin and granzyme B results in apoptosis, the morphological consequences of which are most clearly seen in the nucleus. It is conventionally accepted that perforin acts by perforating the target cell membrane; however, the site and mode of action of granzyme B are unknown. We have addressed this issue using Western blotting, proteolytic assays, and confocal laser scanning microscopy to demonstrate that purified human granzyme B can be taken up in large amounts and bound within nuclei. By contrast, perforin and nongranzyme serine proteases did not undergo nuclear uptake. Both unglycosylated human granzyme B (26 kDa) and that bearing high mannose glycosylation (32 kDa) were internalized and bound within nuclei, but forms greater than 32 kDa with complex carbohydrate addition were excluded. The uptake of granzymes was not dependent on net charge, as nuclei absorbed similar quantities of granzyme B at neutral pH and through a range of basic pHs but did not take up other very basic serine proteases such as the mouse mast cell protease 5. Confocal laser scanning microscopy indicated nuclear and nucleolar accumulation of fluoresceinated granzyme B by isolated nuclei. Measurement of the kinetics of nuclear import using an in vitro nuclear transport assay indicated maximal levels of nuclear accumulation of granzyme about 2.5-fold above those in the cytoplasm and nucleolar accumulation a further 3-4-fold higher. Nuclear and nucleolar accumulation were exceedingly rapid, reaching half-maximal levels within 3.3 and 7.5 min, respectively, implying that nuclear accumulation probably occurs prior to transport to the nucleolus. Our observations may provide a mechanism explaining how aspartate-specific cell death proteases access the nuclear substrate poly(ADP-ribose) polymerase, the cleavage of which is an early event in apoptosis.     

EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis

The Arabidopsis ethylene receptor gene ETR1 and two related genes, ERS1 and ETR2, were identified previously. These three genes encode proteins homologous to the two-component regulators that are widely used for environment sensing in bacteria. Mutations in these genes confer ethylene insensitivity to wild-type plants. Here, we identified two Arabidopsis genes, EIN4 and ERS2, by cross-hybridizing them with ETR2. Sequence analysis showed that they are more closely related to ETR2 than they are to ETR1 or ERS1. EIN4 previously was isolated as a dominant ethylene-insensitive mutant. ERS2 also conferred dominant ethylene insensitivity when certain mutations were introduced into it. Double mutant analysis indicated that ERS2, similar to ETR1, ETR2, ERS1, and EIN4, acts upstream of CTR1. Therefore, EIN4 and ERS2, along with ETR1, ETR2, and ERS1, are members of the ethylene receptor-related gene family of Arabidopsis. RNA expression patterns of members of this gene family suggest that they might have distinct as well as redundant functions in ethylene perception.     

A putative role for larval nematode infection in diarrhoeas of lambs which did not respond to anthelmintic drenches

Attempts to control a summer diarrhoea in grazing Finnish landrace lambs which had been unresponsive to anthelmintics and coccidiostats were made by supplementing them with cupric oxide particles and withdrawing a magnesium-rich mineral, while maintaining parasite control measures. The diarrhoea persisted from July to September and plasma pepsinogen activities were raised, suggesting that the anthelmintic did not prevent abomasal damage; the jejunum of an affected lamb showed lesions of parasitic gastroenteritis. Small responses to cupric oxide particles and larger responses to the withdrawal of magnesium were deceptive, possibly being confounded by differences in parasite challenge. In another experiment Finnish landrace lambs were more susceptible to diarrhoea than Suffolk cross lambs in autumn. The susceptibility was then linked to a strong inhibition of worm egg output and may have been caused by a hypersensitive mucosal response to the larval challenge. Plasma pepsinogen concentrations were again raised in the Finnish landrace lambs and did not decline after treatment with anthelmintic, whereas the concentrations increased later in the Suffolk cross lambs, and were apparently responsive to anthelmintic. The cases of diarrhoea were similar to 'July disease' and may have been caused by continuous nematode infections which were only briefly controlled by drenches. Anthelmintic-unresponsive diarrhoea is the term proposed for the disorder, which may be controllable by devices releasing anthelmintic continuously or by a move to less infected pasture. Faecal egg counts remained low in the condition and were diagnostically misleading.     

Purification and characterization of protein D/E, a putative sperm-binding protein involved in fertilization

We describe a method for the efficient purification of a 32 Kd glycoprotein from rat epididymal tissue. The glycoprotein was purified by gel filtration, ion-exchange, affinity, and reverse phase high pressure liquid chromatography. The highly purified glycoprotein was radiolabeled with an iodinatable, cleavable, photoreactive cross-linking agent, 1-[N-(2-hydrox-5-azidobenzoyl)-2-aminoethyl]-4-(-hydroxysuccini mid yl)-succinate (HAHS). The soluble radiolabeled glycoprotein was bound to washed epididymal spermatozoa in a time-dependent, saturable, and reversible manner. Scatchard analysis demonstrates that there are approximately 3,403 binding sites/spermatozoon. The binding efficiency (Kd) for spermatozoa was approximately 2.0 x 10(-10) M. The function of this glycoprotein was verified by using an in vivo artificial insemination fertilization assay. The fertility rate for control spermatozoa was approximately 53%, but the rate for spermatozoa exposed to polyclonal anti-glycoprotein antibodies was only 5%. These data suggest that the binding of the glycoprotein to the surface of rat spermatozoa is mediated by a receptor-type mechanism and is involved in the fertilization process.