Purification and characterization of guanosine 3',5'-monophosphate-inhibited low K(m) adenosine 3',5'-monophosphate phosphodiesterase from human placental cytosolic fractions.

We previously characterized human placental cytosolic cAMP phosphodiesterase (PDE) and found that two low K(m) cAMP PDE isoforms that were very sensitive to inhibition by cGMP and cilostamide were activated by insulin. As a first step toward understanding the mechanisms by which insulin activates this enzyme, we purified the cGMP-inhibited low K(m) cAMP PDE (cGI-PDE) from human placentas. The enzyme was purified 11,700-fold from a pool of 100,000 x g supernatant fractions of 10-15 placentas by ammonium sulfate precipitation, diethylaminoethyl-cellulose chromatography, and affinity chromatography, using an isothiocyanate derivative of cilostamide (CIT-agarose). The specific activity of the affinity-purified enzyme was 432 +/- 17 nmol/min.mg (mean +/- SD; n = 4). Gel permeation chromatography of the CIT-agarose eluates revealed one protein peak that coincided with PDE activity at an elution position of 135,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this protein peak and CIT-agarose eluates revealed the same patterns, indicating that the purified PDE preparations contained multiple proteins with apparent mol wt of 138K, 83K, 72K, 67K, 63K, and 44K. The 138K form appears to be an intact enzyme; an analogous approximately 135K form has recently been identified in rat adipocyte particulate fractions by specific immunoprecipitation or Western immunoblots. In addition, other smaller forms eluted at 135,000 daltons on gel permeation chromatography, suggesting that, although proteolyzed, they must have been associated by either noncovalent interactions or disulfide bonds. All of the protein bands observed on the sodium dodecyl sulfate-polyacrylamide electrophoresis gel reacted with rabbit antibodies raised against human platelet cGI-PDE. Ten peptides from endoproteinase Lys-C-digests of the affinity-purified placental cGI-PDE were isolated and sequenced; sequences of eight peptides were identical to the deduced amino acid sequences in the C-terminal half of a human heart cGI-PDE cDNA, while those of two peptides were not found in the heart enzyme. The sequences of the eight peptides also matched peptide sequences derived from a purified human platelet cGI-PDE. These results provide evidence that the catalytic C-terminal half domain of the placental insulin-sensitive cGI-PDE shares homology with those of human heart and platelet cGI-PDEs. K(m) and maximum velocity values for cAMP and cGMP were 0.57 microM and 862 nmol/min.mg, and 15 microM and 467 nmol/min.mg, respectively. ED50 values for cGMP, cilostamide, and Ro 20-1724 were 0.12, 0.22, and 120 microM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of insulin-like growth factor-binding proteins produced by cultured fibroblasts from patients with noninsulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus, or obesity.

To evaluate whether the production of insulin-like growth factor-binding proteins (IGFBPs) is altered in various pathological states due to modification of the hormonal milieu, we analyzed patterns of IGFBPs released into conditioned medium during 48-h serum-free culture of early passages of human skin fibroblasts from control subjects and patients with metabolic disorders. IGFBP-2, -3, -4, and -5 were identified in the conditioned medium by immunoblotting or RIA. Compared with those in eight control subjects by ligand blot analysis, the levels of IGFBP-3, -2, and -5 were reduced to 43%, 47%, and 53% in 10 noninsulin-dependent diabetic patients, respectively, whereas the levels of IGFBP-3 and -2 were reduced to 36% and 23%, respectively, in 3 nondiabetic obese patients with impaired glucose tolerance. In 2 insulin-dependent diabetic patients, the level of IGFBP-3 was reduced by 25% and 40%, respectively, and IGFBP-2 was not detectable. In contrast, a similar level of IGFBP-4 was detected in both normal and patient's conditioned media, except in 1 insulin-dependent diabetic patient. These data indicate that fibroblasts derived from patients with metabolic disorders retain their intrinsic characteristics even after they are removed from their in vivo hormonal milieu.

Cyclic nucleotide PDE-3. Quantitation of PDE-3A and -3B mRNAs in rat tissues by RNase protection assay.

Type 3 cyclic nucleotide phosphodiesterase (PDE-3) isoforms exhibit a high affinity ("low K(m)") for cAMP and are specifically inhibited by cGMP and a number of pharmacological agents, which increase myocardial contractility, inhibit platelet aggregation, and increase smooth muscle relaxation. The PDE-3 family consists of at least two isozymes, PDE-3A (cardiac type) and PDE-3B (adipocyte type), with distinct tissue-specific distributions. PDE-3A mRNA is highly expressed in the cardiovascular system, whereas PDE-3B mRNA is primarily expressed in adipocytes and hepatocytes. Toward understanding potential roles of PDE-3 in diabetes mellitus, we have established a specific and sensitive RNase protection assay (RPA) for quantitating PDE-3A and PDE-3B mRNA in rat diabetic models. In fatty Zucker diabetic (ZDF) rats, PDE-3A mRNA, but not PDE-3B mRNA, was expressed in heart, whereas liver and white and brown fat tissues predominantly expressed PDE-3B mRNA. Unexpectedly, PDE-3B mRNA expression was approximately 2.5 times higher than PDE-3A mRNA in aorta from both ZDF and Sprague-Dawley (SD) rats. In contrast, expression levels of PDE-3A mRNA in heart were similar in both species. With this RPA, we were thus able to compare PDE-3A and -3B mRNA levels in different tissues as well as in different rat species.

[Cerebral embolism in systemic lupus erythematosus with Libman-Sacks endocarditis: a case report].

We presented a patient of cerebral embolism caused by Libman-Sacks endocarditis with systemic lupus erythematosus (SLE). This 35-year-old housewife with SLE suffered from abrupt visual disturbance in December 1998. Angiography revealed the occlusion of her right posterior cerebral artery. Transesophageal echocardiography showed the mitral regurgitation and hyperplasia of the anterior mitral valve leaflet without vegetation. In April 1999, she again suffered from sudden onset of transient left hemiparesis and dysphasia. Angiographic findings were unchanged. Transesophageal echocardiographic examination detected vegetation on the anterior mitral valve leaflet and aggravation of the mitral regurgitation. Laboratory examination revealed inactivity of SLE. No bacteria was recovered from repeated blood cultures. We diagnosed that Libman-Sacks vegetation caused cerebral embolism.

[A case of brain infarction of lateral geniculate body showing strange formed scotomas].

A 58-year-old woman suddenly noticed that soccer players disappeared and emerged in right inferior portion of her vision while she was watching soccer game on TV. She was admitted in our hospital on the day 9. Goldman perimeter revealed strange formed hemianopic soctomas which located at right side of her both visual fields. Brain MRI scan showed a tiny infarction in left lateral geniculate body. No abnormalities were found on cerebral angiography and 24 hours holter ECG study. Transesophageal echocardiography and transcranial Doppler study showed the presence of intracardiac right-to-left shunting via atrial septum defect. Paradoxical embolism was considered and oral anticoagulation therapy was started. Her visual defect disappeared by the day 79. Strange formed homonymous hemianopic scotomas were attributable to highly localized small lesion in left anterior choroidal artery territory of lateral geniculate body.

[Paradoxical embolism with Chiari network, subsequently being accompanied by probable incomplete infarction: a case report].

We presented a patient of paradoxical embolism with Chiari network, subsequently being accompanied by probable incomplete infarction. This 21-year-old man suffered from consciousness disorder, aphasia and right hemiparesis, and hospitalized in November 6, 1999. Magnetic resonance imaging showed mixed intensity on T1 and T2-weighted images in part of the areas of the left anterior and middle cerebral arteries. Cerebral angiography revealed the early venous fillings and the capillary blushs. These findings implicated stroke in young adult. Still more transcranial color-flow imaging showed high intensity transient signals with "Chirp" sounds on the left middle cerebral artery. Transesophageal echocardiography detected Chiari network. Chiari network was thought the course of cerebral infarction. Over again 123I-IMP single-photon emission CT findings revealed the marked reduction of his cerebral blood flow comprehensively in the left hemispherium. It was suggested that the recanalization after the paradoxical cerebral embolism had caused incomplete infarction.

[Chronic encapsulated intracerebral hematoma in thalamus with incongruous right homonymous hemianopia: a case report].

We presented a patient with chronic encapsulated intracerebral hematoma. This 49-year-old woman suffered from visual disturbance, and slowly progressive right hemiparesis, sensory disturbance of the right extremities and incongruous right homonymous hemianopia over 2 months. Computed tomography scanning showed high density area and ring enhancement, and magnetic resonance imaging revealed mixed intensity on T1 and T2-weighted images in her left thalamus and internal capsule. Angiographic studies revealed no vascular anomaly or tumor stain. The pathologic pictures indicated well-encapsulated hematoma containing fresh and old hematomas in the left thalamus. Most reported cases of this disease had hematomas in the subcortex and no cases had similar visual disturbance. This report was prepared because this condition is uncommon and may remain unrecognized.

[A case of pharyngeal-cervical-brachial variant of Guillain-Barré syndrome with positive anti-galactocerebroside (Gal-C) IgM antibody].

A 49-year-old man presented with hoarseness, dysphagia, muscle atrophy and weakness of deltoid, trapezius, sternocleidomastoid, rhomboid, anterior serratus, infraspinatus and supraspinatus. Anti-Gal-C IgM antibody was positive in the serum. The other antiganglioside antibodies (GM1, GM2, GM3, GD1a, GD1b, GD3, GT1a, GT1b, GQ1b, GA1, GalNAc-GD1a, GM1b) were negative. Patient contracted pneumonia but whether it was due to mycoplasma was not evident. Plasmapheresis improved his clinical state including a decrease of the antibody. This case was diagnosed pharyngeal-cervical-brachial variant of Guillain-Barré syndrome, and anti-Gal-C antibody seemed to be correlated with the pathogenesis of this syndrome. Gal-C is a major glycolipid of myelin and the cell membrane of the myelin-forming cell (oligodendrocytes and Schwann cells) and is free of specific localization and distribution. The mechanism how the anti-Gal-C IgM antibody induced bulbar paralysis and the symptoms localizing neck and upper limbs remains to be known.

A putative amino acid transporter of the solute carrier 6 family is upregulated by lithium and is required for resistance to lithium toxicity in Drosophila.

Lithium is an efficacious drug for the treatment of mood disorders, and its application is also considered a potential therapy for brain damage. However, the mechanisms underlying lithium's therapeutic action and toxic effects in the nervous system remain largely elusive. Here we report on the use of a versatile genetic model, the fruit fly Drosophila melanogaster, to discover novel molecular components involved in the lithium-responsive neurobiological process. We previously identified CG15088, which encodes a putative nutrient amino acid transporter of the solute carrier 6 (SLC6) family, as one of the genes most significantly upregulated in response to lithium treatment. This gene was the only SLC6 gene induced by lithium, and was thus designated as Lithium-inducible SLC6 transporter or List. Either RNA interference (RNAi)-mediated knockdown or complete deletion of List resulted in a remarkable increase in the susceptibility of adult flies to lithium's toxic effects, whereas transgenic expression of wild-type List significantly suppressed the lithium hypersensitive phenotype of List-deficient flies. Other ions such as sodium, potassium and chloride did not induce List upregulation, nor did they affect the viability of flies with suppressed List expression. These results indicate that lithium's biochemical or physical properties, rather than general osmotic responses, are responsible for the lithium-induced upregulation of List, as well as for the lithium-susceptible phenotype observed in List knockdown flies. Interestingly, flies became significantly more susceptible to lithium toxicity when List RNAi was specifically expressed in glia than when it was expressed in neurons or muscles, which is consistent with potential glial expression of List. These results show that the List transporter confers resistance to lithium toxicity, possibly as a consequence of its amino acid transporter activity in CNS glia. Our results have provided a new avenue of investigation toward a better understanding of the molecular and cellular mechanisms that underlie lithium-responsive neurobiological process.

The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females.

Transient receptor potential (TRP) channels play crucial roles in sensory perception. Expression of the Drosophila painless (pain) gene, a homolog of the mammalian TRPA1/ANKTM1 gene, in the peripheral nervous system is required for avoidance behavior of noxious heat or wasabi. In this study, we report a novel role of the Pain TRP channel expressed in the nervous system in the sexual receptivity in Drosophila virgin females. Compared with wild-type females, pain mutant females copulated with wild-type males significantly earlier. Wild-type males showed comparable courtship latency and courtship index toward wild-type and pain mutant females. Therefore, the early copulation observed in wild-type male and pain mutant female pairs is the result of enhanced sexual receptivity in pain mutant females. Involvement of pain in enhanced female sexual receptivity was confirmed by rescue experiments in which expression of a pain transgene in a pain mutant background restored the female sexual receptivity to the wild-type level. Targeted expression of pain RNA interference (RNAi) in putative cholinergic or GABAergic neurons phenocopied the mutant phenotype of pain females. However, target expression of pain RNAi in dopaminergic neurons did not affect female sexual receptivity. In addition, conditional suppression of neurotransmission in putative GABAergic neurons resulted in a similar enhanced sexual receptivity. Our results suggest that Pain TRP channels expressed in cholinergic and/or GABAergic neurons are involved in female sexual receptivity.

Changes in expression of sensory organ-specific microRNAs in rat dorsal root ganglia in association with mechanical hypersensitivity induced by spinal nerve ligation.

Chronic neuropathic pain caused by peripheral nerve injury is associated with global changes in gene expression in damaged neurons. To understand the molecular mechanisms underlying neuropathic pain, it is essential to elucidate how nerve injury alters gene expression and how the change contributes to the development and maintenance of chronic pain. MicroRNAs are non-protein-coding RNA molecules that regulate gene expression in a wide variety of biological processes mainly at the level of translation. This study investigated the possible involvement of microRNAs in gene regulation relevant to neuropathic pain. The analyses focused on a sensory organ-specific cluster of microRNAs that includes miR-96, -182, and -183. Quantitative real-time polymerase chain reaction (qPCR) analyses confirmed that these microRNAs were highly enriched in the dorsal root ganglion (DRG) of adult rats. Using the L5 spinal nerve ligation (SNL) model of chronic neuropathic pain, we observed a significant reduction in expression of these microRNAs in injured DRG neurons compared to controls. In situ hybridization and immunohistochemical analyses revealed that these microRNAs are expressed in both myelinated (N52 positive) and unmyelinated (IB4 positive) primary afferent neurons. They also revealed that the intracellular distributions of the microRNAs in DRG neurons were dramatically altered in animals with mechanical hypersensitivity. Whereas microRNAs were uniformly distributed within the DRG soma of non-allodynic animals, they were preferentially localized to the periphery of neurons in allodynic animals. The redistribution of microRNAs was associated with changes in the distribution of the stress granule (SG) protein, T-cell intracellular antigen 1 (TIA-1). These data demonstrate that SNL induces changes in expression levels and patterns of miR-96, -182, and -183, implying their possible contribution to chronic neuropathic pain through translational regulation of pain-relevant genes. Moreover, SGs were suggested to be assembled and associated with microRNAs after SNL, which may play a role in modification of microRNA-mediated gene regulation in DRG neurons.

Multiple transcripts for the human cardiac form of the cGMP-inhibited cAMP phosphodiesterase.

cDNAs for two distinct Type III cGMP-inhibited (cGI) cyclic nucleotide phosphodiesterases (PDE), designated cGIP1 and cGIP2, were previously cloned from rat adipose and human cardiac cDNA libraries, respectively. In this study, another cDNA (approximately 4.0 kilobase (kb)) encoding a cGI-PDE of 74 kDa (658 amino acids) was isolated from a human placental cDNA library. The nucleotide sequence of its open reading frame was virtually identical to a corresponding region in the 3' portion of the cardiac cGIP2 cDNA (approximately 7.6 kb) which encoded a approximately 125-kDa cGI-PDE (1141 amino acid). Northern blots and RNase protection assays revealed a prominent 4.4-kb transcript and a 7.6-kb transcript in human placenta. The transcription start site of the 4.4-kb transcript was assigned to cardiac cDNA nucleotide 1292, the putative beginning of exon 3 of the human cGIP2 gene, with a potential translation initiation site 183 bases downstream, as determined by RNase protection assay. The 5'-flanking region of the 4.4-kb transcript exhibited promoter activity in HeLa cells which expressed the 4.4-kb transcript, and contained a TATAA sequence 35 base pairs upstream from the tentative transcription start site. Recombinant cGI-PDEs, expressed in Sf9 cells from the 7.6- and 4.0-kb cDNA, exhibited differences in their subcellular localization and Km for cGMP. Thus, in human tissues, alternative transcription may contribute to generating at least two cGIP2 isoforms, cytosolic and membrane-associated cGI-PDEs with different Km values for cGMP.

Two new monoclonal antibodies against the alpha subunit of the human insulin-like growth factor-I receptor.

Recently, we have reported three monoclonal antibodies (mAbs) against purified human placental insulin-like growth factor (IGF)-I receptors. These antibodies, in contrast to the well-studied mAb alpha IR-3, stimulate binding of IGF-I and IGF-II to the receptor and DNA synthesis as well [Xiong, et al., Proc. Natl. Acad. Sci. U.S.A. 1992(89), 5356]. Here we describe two additional mAbs, 1H7 and 2C8, against the IGF-I receptor that have characteristics different from either alpha IR-3 or our previously reported mAbs. Both 1H7 and 2C8 bind to the alpha subunit of the IGF-I receptor as determined by immunoblotting. MAb 1H7 inhibited the binding of IGF-I and IGF-II to the IGF-I receptor while 2C8 had no effect on the binding of either ligand to the receptor. When their effects on DNA synthesis were examined using NIH 3T3 cells expressing human IGF-I receptors, 1H7 inhibited basal and IGF-I- or IGF-II-stimulated DNA synthesis whereas 2C8 stimulated basal DNA synthesis but provided no synergism in the presence of IGF-I or IGF-II.

Activation of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase during the differentiation of HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate.

We previously reported that the synthesis of NeuAc(alpha 2-3)Gal(beta 1-4)GlcCer (GM3) ganglioside was preferentially enhanced during the differentiation of HL-60 cells into a monocyte/macrophage lineage induced by 12-O-tetradecanoylphorbol-13-O-acetate (TPA). Since exogenously added GM3 ganglioside was shown to be able to induce the differentiation of HL-60 cells into the monocyte/macrophage lineage in a synthetic medium, the functional role of the GM3 ganglioside increase during the differentiation of HL-60 cells has become the subject of much interest. In the present study, we investigated the activity of CMP-NeuAc:lactosylceramide sialyltransferase, which catalyzes the synthesis of GM3 ganglioside from lactosylceramide, in cells undergoing differentiation induced by two different reagents, TPA and 1 alpha,25-dihydroxy-vitamin D3, which induce the differentiation of HL-60 cells into the monocyte/macrophage lineage through different modes of action. We showed that the activation of CMP-NeuAc:lactosylceramide sialyltransferase and the increase in GM3 ganglioside were not related to the differentiated lineage but to the specific action of TPA, i.e. activation of protein kinase C.

Growth-stimulatory monoclonal antibodies against human insulin-like growth factor I receptor.

Monoclonal antibodies (mAbs) against purified human placental insulin-like growth factor I (IGF-I) receptors were prepared and characterized. Three IgG mAbs were specific for the human IGF-I receptor and displayed negligible crossreactivity with the human insulin receptor. They stimulated 125I-labeled IGF-I (125I-IGF-I) or 125I-IGF-II binding to purified human placental IGF-I receptors and to IGF-I receptors expressed in NIH 3T3 cells in contrast to the well-studied mAb alpha IR-3, which inhibits 125I-IGF-I or 125I-IGF-II binding to both forms of IGF-I receptors. The mAbs introduced in this study stimulated DNA synthesis in NIH 3T3 cells expressing human IGF-I receptors approximately 1.5-fold above the basal level and the IGF-I- or IGF-II-stimulated level. In contrast, alpha IR-3 inhibited both basal and IGF-I or IGF-II-stimulated DNA synthesis by approximately 30%. Inhibition of IGF-II-stimulated DNA synthesis by alpha IR-3 was as potent as its inhibition of IGF-I-stimulated DNA synthesis, although IGF-II binding to the IGF-I receptors was not inhibited by IGF-II as potently as was IGF-I. With the purified IGF-I receptors, both inhibitory and stimulatory mAbs were shown to activate autophosphorylation of the IGF-I receptor beta subunit and to induce microaggregation of the receptors. These results suggest that conformational changes resulting from receptor dimerization in the presence of either type of mAb may affect the signal-transducing function of the IGF-I receptor differently. These additional mAbs and alpha IR-3 immunoprecipitated nearly 90% of IGF-I binding activity from Triton X-100-solubilized human placental membranes, indicating that IGF-I receptor reactive with these mAbs is the major form of the IGF-I receptor in human placenta.

Apoptotic proteins Reaper and Grim induce stable inactivation in voltage-gated K+ channels.

Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts. N-terminal sequences of these proteins are highly conserved and are similar to N-terminal inactivation domains of voltage-gated potassium (K+) channels. Synthetic Reaper and Grim N terminus peptides induced fast inactivation of Shaker-type K+ channels when applied to the cytoplasmic side of the channel that was qualitatively similar to the inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K+ channel inactivation correlated with apoptotic activity. Coexpression of Reaper RNA or direct injection of full length Reaper protein caused near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K+ channels.

The purified COOH-terminal domain of the insulin receptor carries activity to stimulate protein kinase activity or autophosphorylation of the beta subunit domain of insulin receptor.

Our previous study using a deletion mutant indicated that the COOH-terminal (CT) domain of the insulin receptor plays important roles in both catalytic efficiency and stability of the receptor kinase (Yan et al., J. Biol. Chem., 268 [1993] 22444). In this study, we purified the CT domain of 98 amino acids from bacterial cells over-expressing the CT domain and examined its effect on insulin and IGF-I receptor protein kinases. The purified CT domain stimulated the kinase activities of purified insulin receptor-transmembrane/cytoplasmic domain (IRTMTPK) and its CT domain-deletion mutant (IRTMTPK delta CT), 3.3-fold and 2.3-fold, respectively, while it was less effective in stimulating the kinase activity of purified IGF-I receptor transmembrane/cytoplasmic domain (IGFIRTMTPK) (1.4-fold). When the effect of the CT domain on autophosphorylation was examined, a marked increase in autophosphorylation was observed only with IRTMTPK delta CT. These results suggest that the CT domain specifically interacts with the insulin receptor cytoplasmic domain, thereby activating the kinase or autophosphorylation activity.

Isolated medial medullary infarction due to vertebral artery dissection.

A 54-year-old man developed left hemiparesis and tactile and deep sensory disturbance following onset of rightside cervical pain. These symptoms resulted from an isolated infarct in the right medial area of the upper medulla oblongata and intracranial vertebral artery (VA) dissection. Atherosclerotic disease of the VA is the most common cause of medial medullary infarction. In past reports of isolated medial medullary infarction, only a few cases involved VA dissection.

Characterization of human placental insulin-like growth factor-I/insulin hybrid receptors by protein microsequencing and purification.

Protein microsequencing of human placental IGF-I receptors purified by immunoaffinity chromatography using IGF-I receptor specific monoclonal antibody revealed amino acid sequences of both IGF-I and insulin receptors. Since this finding indicated the presence of IGF-I/insulin receptor hybrids, hybrid receptors were further purified by immunoaffinity chromatography using insulin receptor specific monoclonal antibody. The molecular size of the nonreduced hybrid receptor was approximately 350K, indicating that the IGF-I and insulin receptor alpha beta halves were disulfide-linked. The ratio of IGF/insulin binding activity of purified hybrid receptors was approximately 25 when measured using tracer amounts of radioactive ligands. 125I-IGF binding to these receptors was inhibited by IGF-I and insulin with IC50s of approximately 2 and approximately 1000 nM, respectively. 125I-Insulin binding to these receptors was similarly inhibited by IGF-I and insulin with IC50 of approximately 3 nM. Autophosphorylation and kinase activities of the hybrid receptor were stimulated by IGF-I more effectively than insulin in a dose-dependent manner. Thus, the present studies indicate that hybrid receptors purified from human placenta have the functional properties of an IGF-I receptor.