Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions between positions 37 and 42

The finger domain deletion mutant of tissue type plasminogen activator (t-PA) has a prolonged half-life in vivo but tends to be accompanied by a decrease in affinity for fibrin. We considered that further refinements in this mutant would reduce the clearance rate and maintain high affinity for fibrin. Two variants, t-PA N37S.S38V G39V R40E.A41S.Q42S and t-PA N37S.S38V G39V R40E.A41F Q42S had a 5-fold increased half-life, but the amount of these variants that bound fibrin was lower than that of wild-type t-PA. To further investigate the effects of these modifications and create a t-PA variant with higher affinity for fibrin, we produced six variants in which residues 37–42 were substituted by more hydophobic amino acids. These variants had various degrees of decreased fibrin binding. Two of them, t-PA N37S.S38VG39VR40I.A41VQ42S and t-PA N37S.S38V.G39V R40I.A41V.Q42L, bound to fibrin stronger than the variants, t-PA N37S.S38VG39VR40E.A41S.Q42S and t-PA N37S.S38VG39VR40E.A41FQ42S, accompanied by a prolonged half-life in vivo.The other objective of this study was to estimate the effects of the amino acid substitution at position 42. Two variants, t-PA N37S.S38VG39VR40E.A41S and t-PA N37S.S38VG39VR40E.A41F had about a 6-fold longer half life in vivo. These data suggest that changes in the pharmacokinetic characteristics of the 37–42 variants are mainly due to modifications at amino acid residues 37–41.     

TRUE Gene Silencing : Unique Gene Silencing by the tRNA Maturase tRNase ZL under the Direction of Small-guide RNA

Recently, signaling by Wnt and its co-receptor, lipoprotein receptor-related protein (LRP) 5, has been suggested to be involved in the regulation of bone formation and bone mass. However, the mechanism by which Wnt signaling achieves this regulation is currently unknown. To assess the functional contribution of Wnt signaling to osteoblastic differentiation, we generated C2C12 cell lines that overexpress either Wnt3a or Wnt5a and treated these cell lines with bone morphogenetic protein (BMP)-2. Expression of bone matrix protein, matrix extracellular phosphoglycoprotein (MEPE), and matrix metalloproteinase (MMP)-13 was induced by BMP-2 in C2C12 cells overexpressing Wnt3a but not Wnt5a. Functional crosstalk between Wnt and BMP signaling during osteoblastic differentiation was also identified. We have now developed a unique system for downregulating gene expression that involves cutting a specific mRNA using the long form of tRNA 3′ processing endoribonuclease (tRNase ZL) under the direction of a small guide RNA (sgRNA). Any potential off-target effects of sgRNA would be expected to be much less serious than those induced by siRNA because the sgRNA targets complementary RNA of only 7–14 nt, a much smaller length of RNA than that targeted by siRNA. We compared the relative efficiency of the sgRNA/tRNase ZL and RNAi methods using an exogenous luciferase gene target. We found that our new method may be more effective at suppressing gene expression. Here, we also show that tRNaseZL, together with sgRNA, can downregulate expression of the endogenous human genes Bcl-2 and GSK-3β by degradation of their mRNAs. These data suggest that the TRUE (tRNase ZL-utilizing efficacious) gene silencing technology has the potential to be used as a therapeutic agent in the treatment of several diseases.     

Mild cognitive impairment and cognitive impairment,no dementia: Part A,concept and diagnosis

Mild cognitive impairment (MCI) and cognitive impairment, no dementia (CIND) are controversial emerging terms that encompass the clinical state between elderly normal cognition and dementia. This article reviews recent work on the classification of MCI and CIND, their prognosis, and diagnostic approaches and presents evidence-based recommendations approved at the meeting of the Third Canadian Consensus Conference on the Diagnosis and Treatment of Dementia (CCCDTD3) held in Montreal in March, 2006. New short tools such as the Montreal Cognitive Assessment are making it easier for family physicians to confidently attach the label of MCI.     

Relationship between perimitral and peritricuspid conduction times

BACKGROUND: Conduction block across the left mitral isthmus (LMI) seems more challenging to achieve and validate compared with the cavotricuspid isthmus (CTI). OBJECTIVE: This study sought to investigate the relationship between peritricuspid and perimitral circuit times in the same patient and to compare the difficulty in achieving the CTI and LMI linear lesions. METHODS: We retrospectively studied 122 consecutive patients (46 paroxysmal and 76 persistent) admitted for atrial fibrillation ablation or subsequent atrial macroreentry who underwent both CTI and LMI ablation. The peritricuspid and perimitral conduction times were measured after validation of bidirectional block across their respective line by pacing from the septal side of the CTI or LMI and recording of the second late potential on the line of block. Atrial dimensions were measured by standard transthoracic echocardiographic techniques. RESULTS: The mean peritricuspid and perimitral times were 180 +/- 35 ms (range 120 to 300) and 189 +/- 42 ms (range 120 to 322), respectively, with a mean difference of 7 +/- 32 ms (-70 to 95). The correlation between both circuit times was highly significant (r = 0.621, P < .001). In 84 patients (68%), the perimitral time was within 30 ms of the peritricuspid time. In the remaining patients, only 12 (10% of the total patients) had a shorter perimitral time compared with peritricuspid time. Radiofrequency energy delivered was significantly longer for LMI (15 +/- 7 min [range 7 to 33]) compared with CTI (7 +/- 4 min [range 3 to 17]) (P = .005). CONCLUSION: The peritricuspid and perimitral circuit times are strongly correlated. In 90% of patients, the perimitral conduction time is within 30 ms or longer than the peritricuspid time. In addition, both circuit times are always > or = than 120 ms. Compared with the left mitral isthmus line, the CTI line is significantly easier to perform.     

Early oculomotor capture by new onsets driven by the contents of working memory

Oculomotor capture can occur automatically in a bottom-up way through the sudden appearance of a new object or in a top-down fashion when a stimulus in the array matches the contents of working memory. However, it is not clear whether or not working memory processing can influence the early stages of oculomotor capture by abrupt onsets. Here we present clear evidence for an early modulation driven by stimulus matches to the contents of working memory in the colour dimension. Interestingly, verbal as well as visual information in working memory influenced the direction of the fastest saccades made in search, saccadic latencies and the curvature of the scan paths made to the search target. This pattern of results arose even though the contents of working memory were detrimental for search, demonstrating an early, automatic top-down mediation of oculomotor onset capture by the contents of working memory.     

Relationship of Condom Use with Other Sexual Risk Behaviors among Selected Japanese Adolescents

This study examines relationships between condom use and age at initiation of sex, the number of sexual partners, and alcohol or drug use before sex among a convenience sample of 2852 Japanese adolescents. We found that nonuse of condoms is related to early initiation of sex, having multiple sexual partners and alcohol or drug use before sex.