Isolation, characterization and synthesis of a novel paradaxin isoform

Recently our laboratory found that tolerance to morphine-induced antinociception could be completely reversed with intracerebroventricular (i.c.v.) administration of a protein kinase A inhibitor, whereas intrathecal (i.t.) administration of the inhibitor produced hyperalgesia in morphine-tolerant mice. In the experiments described here, we sought to characterize further the role of phosphorylation events in supraspinal versus spinal opioid-mediated pain pathways and how such events might be involved in the development of antinociceptive tolerance. Two phosphatase inhibitors were administered centrally to determine whether they affected morphine-induced antinociception in naive or chronically morphine-treated mice. By the i.c.v. route, okadaic acid enhanced morphine-induced antinociception in tolerant mice and produced toxicity by the i.t. route. The calcineurin inhibitor ascomycin had no effect on antinociception following acute or chronic morphine treatment. These results suggest that increased activity of protein phosphatase types 1 and/or 2A in the brain may contribute to the development of morphine tolerance.     

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.     

Molecular cloning and distinct developmental expression pattern of spliced forms of a novel zinc finger gene wiz in the mouse cerebellum

The erbB family of receptor tyrosine kinases are growth factor receptors that are overexpressed or mutated in a large variety of human cancers. Studies of erbB-mediated signal transduction will lead to an understanding of the role played by this family of receptors in normal and transformed cells. In this article, we discuss the contemporary understanding of the structure and function of these receptors, and how these features might be exploited in immunologic strategies of receptor-based growth inhibition. The first part of this article details the structure of erbB receptors as it relates to the process of transformation of cells and the malignant phenotype in human tumors. In the second part of this article, we discuss immunologic approaches to therapy for cancers in which surface-residing erbB receptors are overexpressed or mutated, with an emphasis on studies targeting the p185neu/c-erbB2 oncoprotein. The potential for antireceptor immunity and the evolution of small molecules for receptor-based immunotherapy are discussed. These studies provide a basis for the application of receptor-based strategies of growth inhibition in erbB-expressing human cancers.     

Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern

We have cloned a novel human CC-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1. The isolated cDNA clone (803 bp) shows a single open reading frame of 267-bp coding for 89 amino acid residues; mature AMAC-1 protein is predicted to consist of 69 amino acids with a m.w. of 7855. Sequence alignment and 3D-modeling show the typical structural characteristics of CC-chemokines with special features in the receptor-activating domain. AMAC-1 is most closely related to MIP-1 alpha with a cDNA and protein sequence homology of 55% and 59%, respectively. However, the expression pattern of AMAC-1 is directly opposite to that of MIP-1 alpha. While MIP-1 alpha is induced by classical macrophage mediators such as LPS and is inhibited by IL-4 and glucocorticoids, AMAC-1 is specifically induced in macrophages by alternative macrophage mediators such as IL-4, IL-13, and IL-10. Expression of AMAC-1 is inhibited by IFN-gamma while glucocorticoids exert a slightly positive synergistic effect in combination with IL-4. Peripheral blood monocytes do not express AMAC-1; time course experiments show that monocyte-to-macrophage differentiation is a prerequisite for AMAC-1 expression. Expression of AMAC-1 by granulocyte-macrophage CSF/IL-4-induced, monocyte-derived dendritic cells is complex; in mature adherent dendritic cells, however, only minor AMAC-1 mRNA expression was found. In vivo, AMAC-1 is expressed by alveolar macrophages from healthy persons, smokers, and asthmatic patients. In conclusion, AMAC-1 is a novel CC-chemokine whose expression is induced in alternatively activated macrophages by Th2-associated cytokines; thus, AMAC-1 may be involved in the APC-dependent T cell development in inflammatory and immune reactions.     

Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system

The synucleins are a unique family of small intracellular proteins that have recently attracted considerable attention because of their involvement in human neurodegenerative diseases. We have cloned a new member of the synuclein family called persyn. In contrast to other synucleins, which are presynaptic proteins of CNS neurons, persyn is a cytosolic protein that is expressed predominantly in the cell bodies and axons of primary sensory neurons, sympathetic neurons, and motoneurons. Northern blotting, in situ hybridization, Western blotting, and immunohistochemistry revealed that persyn mRNA and protein are expressed in these neurons from the earliest stages of axonal outgrowth and are maintained at a high level throughout life. Persyn also becomes detectable in evolutionary recent regions of the brain by adulthood.     

Synaptojanin 1: localization on coated endocytic intermediates in nerve terminals and interaction of its 170 kDa isoform with Eps15

This investigation characterized the smooth muscle relaxing effect of a novel nitric oxide (NO)-releasing substance, GEA 3175 (1,2,3,4-oxatriazolium, 3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino], hydroxide inner salt) on guinea-pig trachea. GEA 3175 caused a concentration-dependent relaxation of tracheal smooth muscle precontracted with acetylcholine. This effect was reversed by both okadaic acid, an inhibitor of serine/threonine-specific phosphatases, and iberiotoxin, an inhibitor of Ca2+-activated K+ channels. Furthermore, GEA 3175 had a relaxation potency similar to that of the commonly used NO-donor, S-nitroso-N-acetyl-penicillamine. On the contractile response provoked by electrical field stimulation, GEA 3175 induced a long-lasting relaxation which persisted even after repeated washing. The relaxing effect of GEA 3175 was associated with rises in guanosine 3':5'-cyclic monophosphate (cGMP). In time course studies, cGMP continued to increase with incubation time after stimulation with GEA 3175 and there was a significant elevation of cGMP even after washing. In contrast, incubation with S-nitroso-N-acetyl-penicillamine caused a transient rise in cGMP. The present investigation showed that GEA 3175 evokes long-lasting effects on contractile responses and cGMP levels in guinea-pig trachea. Our results indicate that the relaxing effect of GEA 3175 occurs through a mechanism involving phosphatases and iberiotoxin-sensitive K+ channels.     

Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase

By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSLtes. Due to an addition of amino acids at the NH2-termini, rat and human HSLtes consist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSLadi). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSLadi. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSLadi sequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M(r) approximately 120,000) that exhibited catalytic activity similar to that of HSLadi. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells.     

Cloning, expression and isoform classification of a minor oleosin in sesame oil bodies

The oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosins are present in the oil bodies of diverse angiosperms, and classified as high and low Mr isoforms according to their relative molecular masses in each species. In sesame oil bodies, besides the two ubiquitous oleosin isoforms (17 and 15 kDa), an additional minor oleosin (15.5 kDa) was revealed on Tricine SDS-PAGE. A full-length cDNA fragment was cloned, sequenced and deduced to be a putative oleosin of 15,446 Da. The gene was constructed in a fusion or non-fusion vector and then over-expressed with different efficiency in Escherichia coli. All three oleosins purified from sesame oil bodies were subjected to immunoassaying using antibodies raised against the over-expressed oleosin. The results confirmed that this gene encodes the sesame 15.5 kDa oleosin. Sequence comparisons with other known oleosins revealed that sesame 15.5 kDa oleosin does not represent a new oleosin isoform class but may have been derived through gene duplication and truncation of sesame 17 kDa oleosin, and possesses the minimal structure of the high Mr oleosin isoform. A conserved amphipathic alpha-helix is predicted in sesame 15.5 kDa oleosin, which may imply a potential biological function associated with this isoform.     

Spatially distinct expression of two new cytochrome P450s in leaves of Nepeta racemosa: identification of a trichome-specific isoform

Using a PCR-based approach, two novel cytochrome P450 cDNAs were isolated from a catmint (Nepeta racemosa) leaf cDNA library. The cDNAs (pBSK3C7 and pBSK4C3) were 76.9% identical in their nucleotide sequences, indicating that they are the products of two closely-related genes. A comparison of the sequence of these cDNAs with database sequences indicated that they represent new members of the CYP71 gene family of plant cytochrome P450s. Clone pBSK3C7 contains the full-length coding sequence of a cytochrome P450, whilst pBSK4C3 lacks ca. 6 codons at the 5' end. The cytochromes P450 encoded by these clones were designated CYP71A5 and CYP71A6 (pBSK3C7 and pBSK4C3, respectively). Southern blot analysis indicated that the corresponding genes were present as single copies in the genome of N. racemosa. Northern blot analysis showed that a gene homologous with CYP71A5 was expressed in the related species N. cataria, but no homologue of CYP71A6 was detected in this species. Expression of CYP71A5 in N. racemosa was maximal in flowers, tissues within the apical bud, and young expanded leaves. That of CYP71A6 was maximal in older leaves. Expression of CYP71A5 occurred exclusively in trichomes present on the leaf surfaces, in contrast to that of CYP71A6, which occurred predominantly within the leaf blade tissues.     

Mist1: a novel basic helix-loop-helix transcription factor exhibits a developmentally regulated expression pattern

An equilibrium between positive and negative regulation of immunoreceptor signaling leads to the proper execution of lymphocyte activation. Tyrosine phosphorylation is the initial event in antigen receptor-induced lymphocyte activation. It is generally accepted that protein tyrosine kinases are involved in positive regulation, whereas protein tyrosine phosphatases are important for the negative regulation of tyrosine phosphorylation-dependent processes. However, the interaction between protein tyrosine kinases and protein tyrosine phosphatases is complex. This article discusses the role of two protein tyrosine phosphatases. CD45 and SHP-1, in the regulation of immunoreceptor signaling. SHP-1 acts as a negative regulator for several immunoreceptors, including those for T- and B-cell antigen receptors. The major role of CD45 is in the positive regulation of T- and B-cell antigen receptor signaling.     

Cloning and expression of a novel pH-sensitive two pore domain K+ channel from human kidney

A complementary DNA encoding a novel K+ channel, called TASK-2, was isolated from human kidney and its gene was mapped to chromosome 6p21. TASK-2 has a low sequence similarity to other two pore domain K+ channels, such as TWIK-1, TREK-1, TASK-1, and TRAAK (18-22% of amino acid identity), but a similar topology consisting of four potential membrane-spanning domains. In transfected cells, TASK-2 produces noninactivating, outwardly rectifying K+ currents with activation potential thresholds that closely follow the K+ equilibrium potential. As for the related TASK-1 and TRAAK channels, the outward rectification is lost at high external K+ concentration. The conductance of TASK-2 was estimated to be 14.5 picosiemens in physiological conditions and 59.9 picosiemens in symmetrical conditions with 155 mM K+. TASK-2 currents are blocked by quinine (IC50 = 22 microM) and quinidine (65% of inhibition at 100 microM) but not by the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine, and Cs+. They are only slightly sensitive to Ba2+, with less than 17% of inhibition at 1 mM. As TASK-1, TASK-2 is highly sensitive to external pH in the physiological range. 10% of the maximum current was recorded at pH 6. 5 and 90% at pH 8.8. Unlike all other cloned channels with two pore-forming domains, TASK-2 is essentially absent in the brain. In human and mouse, TASK-2 is mainly expressed in the kidney, where in situ hybridization shows that it is localized in cortical distal tubules and collecting ducts. This localization, as well as its functional properties, suggest that TASK-2 could play an important role in renal K+ transport.