Yoshio Misumi

High-density cDNA filter analysis: a novel approach for large-scale, quantitative analysis of gene expression

In order to analyze the expression profiles of a large number of genes in the tissues (or cells) of interest, and to identify the genes preferentially expressed in the tissues, we have developed a large-scale gene expression analysis system. It is based on the hybridization of the mRNAs from the tissues with a high-density cDNA filter followed by the quantitative measurement of the amount of the hybridized mRNA on each cDNA spot. By employing a high-performance bioimaging analyzer, the system allowed us to compare the expression profiles of thousands of genes (cDNAs) simultaneously with a sensitivity comparable to conventional Northern blotting analysis. By this system (called high-density cDNA filter analysis or HDCFA), the expression profiles of 2505 cloned human brain cDNAs (genes) were monitored. Through the comparison of the expression profiles of these cDNAs in the adult brain, fetal brain and adult liver, about one half of these brain cDNAs (1239 clones) were identified as the candidates which were expressed preferentially in the brain. Among these, 408 and 288 clones were found to be preferentially expressed in the adult and fetal brain, respectively. The results have shown that the system may be widely applicable for analysis of the gene expression profiles of various tissues on a large scale.

Role of the PLC-related, catalytically inactive protein p130 in GABAA receptor function

The protein p130 was isolated from rat brain as an inositol 1,4,5‐trisphosphate‐binding protein with a domain organization similar to that of phospholipase C‐δ1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for γ‐aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABAA receptor‐associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABAA receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA‐induced Cl− current by Zn2+ or diazepam, both of which act at GABAA receptors containing γ subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABAA receptors, especially in response to the agents acting on a γ2 subunit.

Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).

Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes

The effects of brefeldin A on intracellular transport and posttranslational modification of complement C3 (C3) were studied in primary culture of rat hepatocytes. In the control culture C3 was synthesized as a pre‐cursor (pro‐C3), which was processed to the mature form with α‐ and β‐subunits before its discharge into the medium. In the presence of brefeldin A the secretion of C3 was strongly blocked, resulting in accumulation of pro‐C3. However, after a prolonged interval the mature form of C3 was finally secreted. The results indicate that brefeldin A impedes translocation of pro‐C3 to the Golgi complex where pro‐C3 is converted to the mature form, but not its proteolytic processing, in contrast to the effects of monensin and weakly basic amines.

A human Dravet syndrome model from patient induced pluripotent stem cells

BackgroundDravet syndrome is a devastating infantile-onset epilepsy syndrome with cognitive deficits and autistic traits caused by genetic alterations in SCN1A gene encoding the α-subunit of the voltage-gated sodium channel Nav1.1. Disease modeling using patient-derived induced pluripotent stem cells (iPSCs) can be a powerful tool to reproduce this syndrome’s human pathology. However, no such effort has been reported to date. We here report a cellular model for DS that utilizes patient-derived iPSCs.ResultsWe generated iPSCs from a Dravet syndrome patient with a c.4933C>T substitution in SCN1A, which is predicted to result in truncation in the fourth homologous domain of the protein (p.R1645*). Neurons derived from these iPSCs were primarily GABAergic (>50%), although glutamatergic neurons were observed as a minor population (<1%). Current-clamp analyses revealed significant impairment in action potential generation when strong depolarizing currents were injected.ConclusionsOur results indicate a functional decline in Dravet neurons, especially in the GABAergic subtype, which supports previous findings in murine disease models, where loss-of-function in GABAergic inhibition appears to be a main driver in epileptogenesis. Our data indicate that patient-derived iPSCs may serve as a new and powerful research platform for genetic disorders, including the epilepsies.

Domains of the SFL1 protein of yeasts are homologous to Myc oncoproteins or yeast heat-shock transcription factor

We identified a yeast suppressor gene for flocculation (SFL1), which complemented a newly isolated sfl1 mutant. This mutation causes asexual cell aggregation. SFL1 encodes a 767-amino acid protein which has two domains significantly homologous to Myc oncoproteins and the yeast heat shock transcription factor (HSTF). The Myc homologous region in SFL1 overlaps with the conserved region in a series of interesting proteins: MyoD1, Drosophila achaete-scute, twist, daughterless gene products and immunoglobulin enhancer-binding proteins. In addition, the N-terminal region of the SFL1 gene product shows extensive homology to the DNA-binding domain of HSTF. Mutational analysis of SFL1 demonstrates that it is required for normal cell-surface assembly in vegetative growth. We propose that the SFL1 gene product may be a transcription factor which is involved in regulation of the gene(s) related to yeast flocculation.

The interaction of two tethering factors, p115 and COG complex, is required for Golgi integrity.

The vesicle‐tethering protein p115 functions in endoplasmic reticulum–Golgi trafficking. We explored the function of homologous region 2 (HR2) of the p115 head domain that is highly homologous with the yeast counterpart, Uso1p. By expression of p115 mutants in p115 knockdown (KD) cells, we found that deletion of HR2 caused an irregular assembly of the Golgi, which consisted of a cluster of mini‐stacked Golgi fragments, and gathered around microtubule‐organizing center in a microtubule‐dependent manner. Protein interaction analyses revealed that p115 HR2 interacted with Cog2, a subunit of the conserved oligomeric Golgi (COG) complex that is known another putative cis‐Golgi vesicle‐tethering factor. The interaction between p115 and Cog2 was found to be essential for Golgi ribbon reformation after the disruption of the ribbon by p115 KD or brefeldin A treatment and recovery by re‐expression of p115 or drug wash out, respectively. The interaction occurred only in interphase cells and not in mitotic cells. These results strongly suggested that p115 plays an important role in the biogenesis and maintenance of the Golgi by interacting with the COG complex on the cis‐Golgi in vesicular trafficking.

Kv4 Accessory Protein DPPX (DPP6) is a Critical Regulator of Membrane Excitability in Hippocampal CA1 Pyramidal Neurons

A-type K+ currents have unique kinetic and voltage-dependent properties that allow them to finely tune synaptic integration, action potential (AP) shape and firing patterns. In hippocampal CA1 pyramidal neurons, Kv4 channels make up the majority of the somatodendritic A-type current. Studies in heterologous expression systems have shown that Kv4 channels interact with transmembrane dipeptidyl-peptidase-like proteins (DPPLs) to regulate the surface trafficking and biophysical properties of Kv4 channels. To investigate the influence of DPPLs in a native system, we conducted voltage-clamp experiments in patches from CA1 pyramidal neurons expressing short-interfering RNA (siRNA) targeting the DPPL variant known to be expressed in hippocampal pyramidal neurons, DPPX (siDPPX). In accordance with heterologous studies, we found that DPPX downregulation in neurons resulted in depolarizing shifts of the steady-state inactivation and activation curves, a shallower conductance-voltage slope, slowed inactivation, and a delayed recovery from inactivation for A-type currents. We carried out current-clamp experiments to determine the physiological effect of the A-type current modifications by DPPX. Neurons expressing siDPPX exhibited a surprisingly large reduction in subthreshold excitability as measured by a decrease in input resistance, delayed time to AP onset, and an increased AP threshold. Suprathreshold DPPX downregulation resulted in slower AP rise and weaker repolarization. Computer simulations supported our experimental results and demonstrated how DPPX remodeling of A-channel properties can result in opposing sub- and suprathreshold effects on excitability. The Kv4 auxiliary subunit DPPX thus acts to increase neuronal responsiveness and enhance signal precision by advancing AP initiation and accelerating both the rise and repolarization of APs.

Taurine amplifies renal kallikrein and prevents salt-induced hypertension in Dahl rats

Objective To determine whether taurine reduces blood pressure by stimulating the renal kallikrein—kinin system. Methods The effects of taurine on blood pressure, urinary kallikrein activity and renal kallikrein gene expression were investigated in Dahl salt-sensitive (Dahl-S) rats. The specificity of the action of taurine was verified by comparison with the action of β-alanine, a carboxylic analogue of taurine. The effect of co-administration of the specific bradykinin B2 receptor antagonist Hoe 140 was also examined. Results Administration of taurine (3% in drinking water) for 4 weeks retarded the development of salt (4% sodium chloride diet)-induced hypertension. Systolic blood pressure at the end of the experiment was significantly higher in control rats than in taurine-treated rats. Urinary sodium excretion was not decreased by the reduction in blood pressure. The heart weight: body weight ratio was significantly lower, and urinary volume and kallikrein excretion were significantly higher, in taurine-treated rats. Renal kallikrein gene expression at weeks 1 and 4 was higher in taurine-treated rats. Systolic blood pressure 3 and 4 weeks after the administration of β-alanine was slightly, but not significantly, lower than that of untreated rats on a high-salt diet, and was accompanied by a significantly lower body weight. Urinary kallikrein excretion decreased with a high-salt diet regardless of β-alanine administration. Continuous systemic administration of Hoe 140 did not cause any significant alteration in blood pressure in Dahl-S rats that received taurine with a high-salt diet. Taurine also showed a renoprotective effect, as judged by a reduction in proteinuria. Conclusion These results suggest that taurine is an effective antihypertensive agent for salt-induced hypertension. Although taurine activated renal kallikrein, further studies are required to confirm the participation of activated kallikrein in the antihypertensive, cardioprotective and renoprotective effects of taurine.