Shoichi Ishiura

The VNTR polymorphism of the human dopamine transporter (DAT1) gene affects gene expression

The human dopamine transporter (DAT1) gene contains a variable number of tandem repeats (VNTR) in its 3′-untranslated region (UTR). The linkage and association between the VNTR polymorphism of DAT1 and various neuropsychiatric disorders have been reported. We have determined the genomic structure of DAT1 genes containing 7-, 9-, 10-, and 11-repeat alleles and examined the effect of VNTR polymorphism in the 3′-UTR region of DAT1 on gene expression using the luciferase reporter system in COS-7 cells. Luciferase expression was significantly higher when the 3′-UTR of the DAT1 gene contained the 10-repeat allele than when it contained the 7- or 9-repeat alleles. This suggests that VNTR polymorphism affects the expression of the dopamine transporter.

Putative function of ADAM9, ADAM10, and ADAM17 as APP -secretase

Abstract The putative α-secretase cleaves the amyloid precursor protein (APP) of Alzheimer’s disease in the middle of the amyloid β peptide (Aβ) domain. It is generally thought that the α-secretase pathway mitigates Aβ formation in the normal brain. Several studies have suggested that ADAM9, ADAM10, and ADAM17 are candidate α-secretases belonging to the ADAM (a disintegrin and metalloprotease) family, which are membrane-anchored cell surface proteins. In this comparative study of ADAM9, ADAM10, and ADAM17, we examined the physiological role of ADAMs by expressing these ADAMs in COS-7 cells, and both “constitutive” and “regulated” α-secretase activities of these ADAMs were determined. We tried to suppress the expression of these ADAMs in human glioblastoma A172 cells, which contain large amounts of endogenous α-secretase, by lipofection of the double-stranded RNA (dsRNA) encoding each of these ADAMs. The results indicate that ADAM9, ADAM10, and ADAM17 catalyze α-secretory cleavage and therefore act as α-secretases in A172 cells. This is the first report that to suggest the endogenous α-secretase is composed of several ADAM enzymes.

Pathophysiology of muscle fiber necrosis induced by bupivacaine hydrochloride (Marcaine).

SummaryA single direct injection of a local anesthetic, 0.5% bupivacaine hydrochloride (BPVC) (Marcaine), into rat soleus and extensor digitorum longus (EDL) muscles produced massive fiber necrosis with extensive phagocytosis followed by rapid regeneration, predominantly in the soleus. Since the sarcoplasmic reticulum (SR) was functionally disturbed by BPVC administration as confirmed by an in vitro study, the sarcolemmal lysis seen in the early phase of degeneration was not assumed to simply results from direct damage to the plasma membrane caused by BPVC. The extracellular fluid containing a high concentration of calcium (Ca) ions then permeated into the sarcoplasm through the defective membrane resulting in hypercontracted myofibrils. Selective damage to the Z-line, an early sign of muscle degeneration, was shown by electron microscopy and SDS gel electrophoresis (preferential loss of α-actinin). Administration of leupeptin, a thiol protease inhibitor, proved to be ineffective in inhibiting the necrotic process, because the BPVC induced muscle fiber breakdown was probably too acute and fulminant to demonstrate the inhibitory effect upon the degenerative process. Well preserved satellite cells, peripheral nerves, and acetylcholinesterase activity, and the absence of fibrous tissue proliferation in this system may be responsible for the extremely rapid regeneration with complete muscle fiber type differentiation. Since the sequence of fiber breakdown induced by BPVC administration was similar to that of progressive muscular dystrophy, this chemical will be one of the most useful tools for studying the pathophysiology of fiber necrosis and regeneration in diseased muscle.

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

Abstract: In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7‐8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 μM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N‐methoxysuccinyl‐Ala‐Ala‐Pro‐Val‐methyl chloride, eluted at a position corresponding to a molecular mass of ∼ 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase‐like protease. This suggestion was confirmed by the finding that the 25‐kDa protein band was stained with anti‐elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopoly‐saccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.

Mosaic Expression of Dystrophin in Symptomatic Carriers of Duchenne's Muscular Dystrophy

A deficiency of the protein dystrophin is known to be the cause of Duchennes muscular dystrophy. To examine the expression of dystrophin in symptomatic female carriers of this X-linked recessive disorder, we performed immunohistochemical studies on muscle-biopsy specimens from three such carriers, using an antiserum raised against a synthetic peptide fragment of dystrophin. In all three carriers, most individual muscle fibers reacted either strongly or not at all to the antiserum for dystrophin; only 2 to 8 percent of fibers showed partial immunostaining. This mosaic staining pattern was present on both cross-sectional and longitudinal muscle specimens. Although the mosaic pattern was seen in all fiber types, more than 80 percent of type 2B and 2C fibers from two of the carriers did not react with the antiserum. Similar studies in nine normal subjects showed consistently strong staining of all muscle fibers. No muscle fibers from 31 patients with Duchennes muscular dystrophy reacted with the antiserum. We conclude that symptomatic carriers of Duchennes muscular dystrophy can be identified by a distinct mosaic pattern in the immunohistochemical staining of the surface membrane of skeletal-muscle specimens. This finding may have practical implications for genetic counseling, although it remains to be shown whether the same staining pattern will be found in muscle specimens from asymptomatic carriers of Duchennes muscular dystrophy.

Minireview: Calcium-dependent proteolysis in living cells

Abstract Calcium ion, one of the second messengers in living organisms, has various functions including the ability to enhance intracellular proteolysis. This calcium-dependent proteolysis occurs in the cytosol or membrane rather than in the lysosome. Its mode of action is very wide, including cleavage of hormone receptors, activation of regulatory enzymes and limited proteolysis of the cytoskeletal structure. Although contradictory, the biochemical evidence implies a specified regulatory function of it in the cell. The activation mechanism of a purified calcium-dependent proteinase ( EC 3.4.22.- ) is also discussed.

Dystrophin-related protein in the fetal and denervated skeletal muscles of normal and mdx mice

The amino acid sequence of the polyclonal antibodies we developed against the carboxyl terminus of the dystrophin-related protein, the putative gene product of B3 cDNA, had no homologous sequence to the dystrophin molecule except for two amino acids located at its ends for immunization. By immunohistochemical examination in C57B1/10ScSn and C57B1/10ScSn-mdx mice we found that the DRP was expressed on the surface membrane of fetal muscle fibers, was assembled at the neuromuscular junctions of the mature muscle fibers, and reappeared on the surface membrane of muscle fibers after denervation. Its localization was similar to that of the acetylcholine receptor, suggesting that DRP is one of the cytoskeletons which organize and stabilize the cytoplasmic domain of the acetylcholine receptor.

The protease activity of a calpain-like cysteine protease in Saccharomyces cerevisiae is required for alkaline adaptation and sporulation

Saccharomyces cerevisiae has only one putative gene (designated CPL1) for a cysteine protease with a protease domain similar to that of calpain. This gene product shows significant sequence similarity to PalBp, a fungal (Emericella nidulans) calpain-like protease that is responsible for adaptation under alkaline conditions, both in the protease domain and the domain following the protease domain. CPL1 disruptant strains show impaired growth at alkaline pH, but no obvious growth defects under acidic pH conditions. This phenotype is complemented by the wild-type CPL1 gene, and its protease activity is essential for complementation. Disruption of CPL1 also causes reduced sporulation efficiency and promotes the degradation of the transcription factor Rim101p, which is involved in the sporulation pathway and has been shown to accumulate in a C-terminally truncated, active form under alkaline conditions. Furthermore, expression of the C-terminally truncated Rim101p suppressed the alkaline sensitivity associated with CPL1 disruption. These results indicate that a calpain-like cysteine protease, Cpl1p, plays an important role in alkaline adaptation and sporulation processes, via regulation of the turnover and processing of the transcription factor Rim101p.

In vivo protection of a water-soluble derivative of vitamin E, Trolox, against methylmercury-intoxication in the rat

Methylmercury (MeHg) is a well-known neurotoxicant. MeHg-intoxication causes a disturbance in mitochondrial energy metabolism in skeletal muscle and apoptosis in cerebellum. We report the first in vivo effectiveness of antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carhoxylic acid), a water soluble vitamin E analog, against the MeHg-induced cellular responses. Treatment with Trolox (6-hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic acid) clearly protects MeHg-treated rat skeletal muscle against the decrease in mitochondrial electron transport system enzyme activities despite the retention of MeHg. Tdt-mediated dUTP nick-end-labeling method clarified that Trolox is effective for protecting cerebellum from MeHg-induced apoptosis. These data indicate that MeHg-mediated oxidative stress plays an important role in the in vivo pathological process of MeHg intoxication. Trolox may prevent some of clinical manifestations of MeHg-intoxication in humans.

A secreted form of human ADAM9 has an α-secretase activity for APP ☆

Abstract ADAM9 (MDC9, meltrin γ) is a member of the ADAM family of metalloproteases, which play important roles in cell–cell fusion, intracellular signaling, and other cellular functions. Here we cloned a novel form of human ADAM9, designated hADAM9s (s for short), which lacks the carboxyl-terminus. Human ADAM9s was found to be secreted from transfected COS cells. RT-PCR analysis demonstrated that the mRNA for hADAM9s is expressed in human brain, liver, heart, kidney, lung, and trachea. When hADAM9s was co-expressed in COS cells with APP and treated with phorbol ester, the APP was digested exclusively at the α-secretory site. These results suggest that hADAM9s has an α-secretase-like activity for APP. Non-amyloidgenic cleavage of APP may occur at the plasma membrane. Our new results support a new therapeutic strategy to decrease in the Aβ content by directly activating ADAM9 in the extracellular space.