Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris

The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A. G. Darvill, and P. Albersheim, Plant J. 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation. Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, and Alternaria solani. No evident enhanced resistance, compared with the resistance of untransformed plants, was observed. The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana plants were tested with several fungal polygalacturonases (PGs). PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P. vulgaris (bulk bean PGIP). Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. Moreover, PGIP-1 expressed in tomato and N. benthamiana had only a limited capacity to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was used to separate PGIP proteins present in P. vulgaris extracts. A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F. moniliforme PGs. Our data show that PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.     

Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types

The type I inositol 1,4,5-trisphosphate (InsP3) receptor can be rapidly depleted from cells during stimulation of phosphoinositide hydrolysis because its degradation is accelerated (Wojcikiewicz, R. J. H., Furuichi, T., Nakade, S., Mikoshiba, K., and Nahorski, S. R. (1994) J. Biol. Chem. 269, 7963-7969). The present study examines the regulatory properties of type II and III InsP3 receptors. Initially, the relative abundance of InsP3 receptors was defined in a range of cell types by quantitative immunoblotting. These studies showed that the proportions in which type I, II, and III InsP3 receptors are expressed differs greatly and that some cells (for example, AR4-2J rat pancreatoma cells) express all three receptors. Analysis of the effects of cholecystokinin and bombesin on AR4-2J cells showed that each of the InsP3 receptors could be down-regulated during activation of phosphoinositide hydrolysis, but that depletion of the type II receptor was limited. Such a discrepancy was also seen in rat cerebellar granule cells and was found to result from the type II receptor being relatively resistant to degradation. In conclusion, type I, II, and III receptors can all be down-regulated, but with different characteristics. As the relative abundance of InsP3 receptors is extremely variable, the extent to which activation of the down-regulatory process alters intracellular signaling will vary depending on which InsP3 receptors are expressed.     

MMP and TIMP expression pattern in pleural effusions of different origins

The matrix metalloproteinases (MMP) are proteolytic enzymes that are essentially involved in the turnover of the extracellular matrix (ECM). Their activity is counterbalanced by specific antagonists, the tissue inhibitors of metalloproteinases (TIMP). In this study, we sought to analyze the expression of MMP and TIMP isoforms in pleural effusions from 88 patients. We compared MMP and TIMP isoform expression in transudates (n = 21) and exudates (n = 67), the latter divided into exudates of paraneoplastic (n = 46) or parainfectious (n = 21) origin. Zymographic and Western blot analyses revealed constant expression of interstitial collagenase (MMP-1), gelatinase-A (MMP-2), and TIMP-1 in all 88 samples. In contrast, analyses of gelatinase-B (MMP-9) demonstrated a specific expression pattern, with high expression in exudates and lack of expression in transudates. Neutrophil collagenase (MMP-8) was detected in trace amounts, and correlated with the number of neutrophils in the effusion. Low levels of TIMP-2 were detected only in exudates and not in transudates. Quantitative analysis of the expression ratio of gelatinase-B to gelatinase-A revealed statistically significant differences between effusions of different origin. The ratio was highest in exudates of paraneoplastic origin and lowest in transudates. Our data thus suggest that interstitial collagenase, gelatinase-A, and TIMP-1 play a role in homeostasis of the pleural space in vivo as constitutively expressed proteins, whereas gelatinase-B and TIMP-2 expression are induced in specific disease states. These observations contribute to the understanding of the pathophysiology of pleural effusions, and may help to characterize and possibly distinguish effusions of different origin.     

Effect of temporal position, proportional variance, and proportional duration on decision weights in temporal pattern discrimination

Two experiments investigated how listeners allocate their attention to different segments of a temporal pattern. The experiments allowed a direct test of the predictions of the Proportion of Total Duration (PTD) rule and the Component Relative Entropy (CoRE) model. Listeners had to decide whether two sequences of nine tones had the same or different temporal patterns (tone duration = 25 ms, tone frequency = 1000 Hz). A sequence's temporal pattern was determined by the time intervals between each tone's offset and the next tone's onset. On same trials, the time intervals at corresponding temporal positions in the two sequences were identical. On different trials, the corresponding time intervals were randomly varied. Listener attention to different temporal positions within a sequence was assessed by calculating the decision weights at each position. The results supported the CoRE model and were inconsistent with the PTD rule. Manipulating the mean of the time intervals within the sequence had no consistent effect on the pattern of weights (or on overall performance), indicating that listener attention was not affected by either the proportion of total duration or the perceptual salience of a longer or shorter time interval. However, manipulating the variance of the time intervals had a significant effect: the highest weight was given to the highest variance segment. This weighting strategy leads to better performance because higher variance segments are more diagnostic of whether the sequences are the same or different.     

Synaptojanin 2, a novel synaptojanin isoform with a distinct targeting domain and expression pattern

Synaptojanin (synaptojanin 1) is a recently identified inositol 5'-phosphatase, which is highly enriched in nerve terminals and is implicated in synaptic vesicle recycling. It is composed of three domains: an amino-terminal SacI homology region, a central inositol 5'-phosphatase homology region, and a carboxyl-terminal proline-rich region. We have now identified and characterized a novel form of synaptojanin, synaptojanin 2, which has a broader tissue distribution. Synaptojanin 2 cDNA from rat brain library encodes a protein of 1,248 amino acids with a predicted Mr of 138,268. The two synaptojanin isoforms share 57.2 and 53.8% amino acid identity in their SacI and phosphatase domains, respectively. In marked contrast, their carboxyl-terminal proline-rich regions bear little homology. Expression of synaptojanin 2 in COS7 cells produced a 140-kDa protein with inositol 5'-phosphatase actvity. Protein binding assays demonstrated that among the major src homology 3-proteins known to bind to the proline-rich region of synaptojanin 1, Grb2, amphiphysin, and members of SH3p4/8/13 protein family, only Grb2 bound to that of synaptojanin 2. Furthermore, subcellular fractionation studies in transfected Chinese hamster ovary cells revealed that synaptojanin 2 was predominantly associated with the particulate fraction while synaptojanin 1 was mainly localized in the soluble fraction. This observation suggests that the proline-rich regions of synaptojanins 1 and 2 are implicated in different protein-protein interactions and direct the two isoforms to different subcellular compartments. Our results demonstrate the presence of a family of synaptojanin-type inositol 5'-phosphatases with different tissue and subcellular distributions, which may be involved in distinct membrane trafficking and signal transduction pathways in mammalian cells.     

Arrestin and phosducin are expressed in a small number of brain cells

Retinal photoreceptor rods and pinealocytes contain well-characterized proteins such as arrestin and phosducin whose expression is highly restricted to these cell types. Transgenic mice having a LacZ gene under the control of an arrestin promoter expressed beta-galactosidase (beta-Gal) in the photoreceptor rods and pinealocytes. In addition, it was expressed in very small numbers of discrete cells in the habenular commissura, amygdala, ventral tegmental area and superior colliculus of the brain. Immunocytochemical studies with antibody probes revealed that high level of arrestin and phosducin were also found in the same cell types. Furthermore melatonin was found in those cells of the habenula commissura. The results indicate that novel cell types are present in the brain tissues. Since high levels of arrestin and phosducin expression are generally restricted to photoreceptor rod cells and pinealocytes, these data suggest that certain brain cells may have functions similar to pinealocytes.     

Structure and expression of a novel human FGF, FGF-19, expressed in the fetal brain

Penetration of fluconazole into female genital tissues was examined. Fluconazole was administered orally at a dose of 150 mg to patients undergoing total abdominal hysterectomy 1 to 151 h prior to surgery. During surgery, blood, uterus, ovary, and oviduct were sampled. Fluconazole concentrations in each tissue were determined by high-performance liquid chromatography. The peak concentrations in serum reached approximately 6.1 microg/ml 1.0 h after a drip infusion was begun. At each time after the infusion, the concentrations in portio vaginalis, cervix uteri, myometrium, endometrium, ovary, and oviduct were higher than those in the serum: the peaks in the tissues ranged from 6.4 to 9.5 microg/g around 1.0 h after the drip infusion was begun. Thus, the levels of penentration of fluconazole into gynecological tissues appeared to be similar to or slightly above those in serum samples. Fluconazole can rapidly penetrate from plasma into the female genital organs, supporting high efficacy of fluconazole against fungal infections in the field of gynecology.     

Resistance to tomato yellow leaf curl geminivirus in Nicotiana benthamiana plants transformed with a truncated viral C1 gene

The C1 gene of tomato yellow leaf curl geminivirus (TYLCV) encodes a multifunctional protein (Rep) involved in replication. A truncated form of this gene, capable of expressing the N-terminal 210 amino acids (aa) of the Rep protein, was cloned under the control of the CaMV 35S promoter and introduced into Nicotiana benthamiana using Agrobacterium tumefaciens. The same sequence was also cloned in antisense orientation. When self-pollinated progeny of 19 primary transformants were tested for resistance to TYLCV by agroinoculation, some plants proved to be resistant, particularly in the sense lines. Two such lines were further studied. The presence of the transgene was verified and its expression was followed at intervals. All plants that were resistant to TYLCV at 4 weeks postinoculation (wpi) contained detectable amounts of transgenic mRNA and protein at the time of infection. Resistance was overcome in a few plants at 9 wpi, and in most at 15 wpi. Infection of leaf discs derived from transgenic plants showed that expression of the transgene correlated with a substantial reduction of viral DNA replication. Cotransfections of tobacco protoplasts demonstrated that inhibition of viral DNA replication requires expression of the truncated Rep protein and suggested that the small ORF C4, also present in our construct, plays no role in the resistance observed. The results obtained using both transient and stable gene expression systems show that the expression of the N-terminal 210 aa of the TYLCV Rep protein efficiently interferes with virus infection.     

Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes

Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H', and F. These proteins share epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H' is 96%, between H and F 78%, and between H' and F 75%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used for ribohomopolymer binding studies. Each qRRM repeat bound poly(rG), while only the NH2-terminal qRRM bound poly(rC) and poly(rU). None of the repeats bound detectable amounts of poly(rA). The expression levels of hnRNPs H and F were differentially regulated in pairs of normal and transformed fibroblasts and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term treatment with 4 beta-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4 beta-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H', and F were chromosome-mapped to 5q35.3 (HNRPH1), 6q25.3-q26, and/or Xq22 (HNRPH2) and 10q11.21-q11.22 (HNRPF), respectively.     

Outside-In signaling of soluble and solid-phase fibrinogen through integrin alphaIIbbeta3 is different and cooperative with each other in a megakaryoblastic leukemia cell line, CMK

The function and the outside-in signaling pathways of alphaIIbbeta3 were examined in relation to cell adhesion using a megakaryoblastic leukemia cell line, CMK. After 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, the cells adhered to the culture plate and underwent megakaryocytic differentiation with expression of alphaIIbbeta3. Binding of soluble fibrinogen to the cells via alphaIIbbeta3 was dependent on cell adhesion. Cell detaching reduced the affinity of this integrin for soluble fibrinogen, although its surface expression was almost unchanged. In contrast, detached cells became tightly adherent to the fibrinogen-coated plate (solid-phase fibrinogen). The same ligand, fibrinogen, present either in soluble or solid-phase form, triggered differential signaling pathways mediated by alphaIIbbeta3. By the stimulation with soluble fibrinogen, Syk was tyrosine-phosphorylated but FAK was dephosphorylated, whereas solid-phase fibrinogen promptly caused tyrosine phosphorylation of FAK followed by delayed phosphorylation of Syk. In addition, the binding of soluble fibrinogen to the cells adherent to fibrinogen-coated plate resulted in tyrosine phosphorylation of integrin beta3 and a complex formation of integrin beta3 with Syk. This implies the cooperation of both soluble and solid-phase fibrinogen-mediated signaling pathways.     

BCR-ABL oncoprotein is expressed by platelets from CML patients and associated with a special pattern of CrkL phosphorylation

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR-ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR-ABL mRNA and with maximal platelet counts ranging from 149 to 3069 x 10(9)/l. P210BCR-ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph-positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR-ABL-negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p210BCR-ABL-positive platelets lysates with anti-CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR-ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR-ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR-ABL-mediated CrkL tyrosine phosphorylation.     

Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes

A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain reaction using primers based on the 5'- and 3'-end sequences of two rat pseudogenes, CYP2J3P1 and CYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolized arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 degrees C). Immunoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model, 20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 +/- 6% of base-line contractile function. The addition of 5 microM 11, 12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in recovery of cardiac contractility (69 +/- 5% of base line, p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11, 12-epoxyeicosatrienoic acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.