Osteoinduction by bone morphogenetic protein 2-expressing adenoviral vector: application of biomaterial to mask the host immune response

We constructed a human bone morphogenetic protein 2 (BMP-2)-expressing adenoviral vector, AxCABMP-2, which showed osteoinduction in immunosuppressed rats. In immunocompetent rats, new bone was not induced, because of the rapid elimination of transduced cells. Biomaterials such as collagen can be used as carriers for the delivery of DNA vectors, allowing prolonged expression of plasmid DNA in normal animals. We evaluated osteoinduction with AxCABMP-2 and atelopeptide type I collagen in immunocompetent rats. Collagen plus AxCABMP-2 (BMP group), collagen plus AxCALacZ (LacZ group), or collagen alone (CL group) was implanted into calf muscle pouches in immunocompetent rats, or AxCABMP-2 alone (injection group) was injected into the calf muscle. On days 3, 7, 14, and 21 after treatment, osteoinduction was evaluated. In the BMP group, bone formation was not observed on days 3 and 7. On day 14, radiographic formation was seen, but little bone formation was detected histologically. On day 21, new bone formation was observed both radiologically and histologically. In the other groups, osteoinduction was not found at any time. Immunohistochemical analysis on days 3 and 7 revealed decreased immunogenicity in the BMP group compared with the injection group. These findings suggested that collagen was an effective masking material for our vector.     

Preparation of PEG-conjugated fullerene containing Gd3+ ions for photodynamic therapy.

A novel photosensitizer with magnetic resonance imaging (MRI) activity was designed from fullerene (C(60)) for efficient photodynamic therapy (PDT) of tumor. After chemical conjugation of polyethylene glycol (PEG) to C(60) (C(60)-PEG), diethylenetriaminepentaacetic acid (DTPA) was subsequently introduced to the terminal group of PEG to prepare PEG-conjugated C(60) (C(60)-PEG-DTPA). The C(60)-PEG-DTPA was mixed with gadolinium acetate solution to obtain Gd(3+)-chelated C(60)-PEG (C(60)-PEG-Gd). Following intravenous injection of C(60)-PEG-Gd into tumor-bearing mice, the PDT anti-tumor effect and the MRI tumor imaging were evaluated. The similar O(2)(*-)generation was observed with or without Gd(3+) chelation upon light irradiation. Both of the C(60)-PEG-Gd and Magnevist(R) aqueous solutions exhibited a similar MRI activity. When intravenously injected into tumor-bearing mice, the C(60)-PEG-Gd maintained an enhanced MRI signal at the tumor tissue for a longer time period than Magnevist(R). Injection of C(60)-PEG-Gd plus light irradiation showed significant tumor PDT effect although the effect depended on the timing of light irradiation. The PDT efficacy of C(60)-PEG-Gd was observed at the time when the tumor accumulation was detected by the enhanced intensity of MRI signal. This therapeutic and diagnostic hybrid system is a promising tool to enhance the PDT efficacy for tumor.     

Simple enzymatic method for determining 1,5-anhydro-D-glucitol in plasma for diagnosis of diabetes mellitus

We have developed a simple method for determination of 1,5-anhydro-D-glucitol in plasma, based on use of pyranose oxidase (EC 1.1.3.10), an enzyme with specificity toward pyranoid compounds such as 1,5-anhydro-D-glucitol and glucose. Plasma samples deproteinized with trichloroacetic acid are passed through a two-layer mini-column packed with strongly basic anion (OH- form, the upper layer) and strongly acidic cation (H+ form, the lower layer) exchange resins. 1,5-Anhydro-D-glucitol is efficiently recovered in the flow-through fraction, which is almost devoid of other sugars that are sensitive to pyranose oxidase. The hydrogen peroxide formed in the enzymatic oxidation of 1,5-anhydro-D-glucitol is detected by a standard method utilizing an enzymatic color-developing system. The overall assay system is highly specific for 1,5-anhydro-D-glucitol. The correlation between results obtained in the present method (x) and in the gas-liquid chromatographic (GLC) method (y) was: y = 1.062x-0.293 mg/L (r = 0.997, n = 49, Sxy = 10.78 mg/L). Compared with GLC, our method is simpler in the sample treatment step and quicker in the measuring step. The precisions of the two methods are comparable.     

Effects of plasma infusion on plasma lipids, apoproteins and plasma enzyme activities in familial lecithin: cholesterol acyltransferase deficiency

Abstract. The siblings presented here are the third family found in Japan with familial LCAT deficiency. Their post-heparin plasma lipoprotein lipase and hepatic triglyceride lipase activities were measured selectively by an immunochemical method. Plasma triglyceride levels were elevated, and post-heparin plasma lipoprotein lipase was decreased only in a patient with nephropathy, while hepatic triglyceride lipase activities were within reference limits in both patients. The plasma concentrations of apo A-I, apo A-II, and apo B were reduced in both patients. On the other hand, the plasma concentration of apo E was markedly increased. Enzyme replacement therapy by plasma transfusion in the propositus resulted in marked improvement of deranged compositions of triglyceride-rich lipoproteins. Also, improvement of the plasma apo E concentration was demonstrated, while the improvement of post-heparin lipase did not occur. These results suggest that LCAT may play an important physiological role in triglyceride metabolism as well as in cholesterol metabolism.     

Comparison of cervical spinal canal diameter between younger and elder generations of Japanese

Background  

Cervical myelopathy is more common among Japanese than Westerners. The shorter anteroposterior diameter of the cervical spinal canals (AP diameter) is its probable cause. In recent years, builds of younger Japanese have become larger and been approaching those of Westerners. The purpose of this study was to investigate whether the cervical spinal canal had enlarged in the younger Japanese as well as any cross-sectional improvement in their builds.     

Effects of aripiprazole and the Taq1A polymorphism in the dopamine D2 receptor gene on the clinical response and plasma monoamine metabolites level during the acute phase of schizophrenia

The Taq1A polymorphism in the dopamine D2 receptor (DRD2) gene could be related to the response to antipsychotics. We examined the effects of the Taq1A polymorphism on the plasma monoamine metabolites during the treatment of schizophrenia with aripiprazole, a DRD2 partial agonist. Thirty Japanese patients with schizophrenia were treated with aripiprazole for 6 weeks. We measured plasma levels of homovanillic acid (pHVA) and 3-methoxy-4hydroxyphenylglycol (pMHPG) before and after treatment. The Taq1A polymorphism was genotyped with polymerase chain reaction. Aripiprazole improved the acute symptoms of schizophrenia and decreased pHVA in responders (P = 0.023) but not in nonresponders (P = 0.28). Although A1 allele carriers showed a tendency to respond to aripiprazole (61.5%) compared to A1 allele noncarriers (29.4%) (P = 0.078), there was not statistically significant difference in the response between the 2 genotype groups. There were significant effect for response (P = 0.013) and genotype × response interaction (P = 0.043) on the change of pHVA. The changes of pHVA differ between responders and nonresponders in A1 allele carriers but not in A1 allele noncarriers. There were no genotype or response effects or genotype × response interaction on the changes of the plasma levels of 3-methoxy-4hydroxyphenylglycol. Our preliminary results suggest that Taq1A polymorphism may be partly associated with changes in pHVA during acute schizophrenia.     

Effects of methylmercury exposure on neuronal differentiation of mouse and human embryonic stem cells

Sulphur mustard (SM) is a blistering agent that causes debilitating damage to the skin, eyes and respiratory system. In cases of severe exposure, immunodepletion can occur as well as death, due to secondary infections. The toxicity of SM is thought to be mediated in part by the alkylation of nucleic acids and proteins, although the exact mechanisms are not clear. In addition, although the first known use of SM was in military conflict nearly 100 years ago, there are still no effective treatments or preventative measures. In order to develop treatments it is necessary to have a detailed understanding of the cellular biochemical changes induced by SM as well as information on the mechanisms that cells employ to protect against SM toxicity. We have previously demonstrated that the homologous recombination (HR) DNA repair pathway promotes cell survival after SM. This study investigated the role of other DNA repair pathways in the cellular response to SM, specifically base excision repair (BER), nucleotide excision repair (NER) and non-homologous end joining (NHEJ) as well as studying the activation and regulation of DNA damage signalling pathways. Our data confirmed that HR is the major repair pathway protecting against acute SM toxicity, with NER and NHEJ also contributing to cell survival. In addition, this study demonstrated the dose- and time-dependent activation of DNA damage signalling pathways after SM in human TK6 lymphoblastoid cells, in particular the phosphorylation of CHK1, CHK2 and p53. These phosphorylation events were orchestrated by a combination of the ATM and ATR protein kinases.     

Comparisons of insulin related parameters in commercial-type chicks: Evidence for insulin resistance in broiler chicks

The aim of this study is to elucidate whether insulin acts differentially within the central nervous system (CNS) of two types of commercial chicks to control ingestive behavior. Male layer and broiler chicks (4-day-old) were intracerebroventricularly (ICV) injected with saline or insulin under satiated and starved conditions. Feed intake was measured at 30, 60 and 120 min after treatment. Secondly, blood and hypothalamus were collected from both chick types under ad libitum feeding and fasting for 24 h. Plasma insulin concentration was measured by time-resolved fluoro-immunoassay. Hypothalamic insulin receptor mRNA expression levels were measured by quantitative RT-PCR. The ICV injection of insulin significantly inhibited feed consumption in layer chicks when compared with saline (P < 0.05), but not broiler chicks (P > 0.1). Plasma insulin concentration of both chick types significantly decreased following 24 h of fasting, while insulin concentrations in the broiler chicks were significantly higher compared to the layers fed under ad libitum conditions. Hypothalamic insulin receptor mRNA expression levels were significantly lower (P < 0.05) in broiler chicks than in layer ones under ad libitum feeding. Feed deprivation significantly decreased insulin receptor mRNA levels in layer chicks (P < 0.01), but not in broiler chicks (P > 0.1). Moreover, plasma insulin concentrations correlated negatively with hypothalamic insulin receptor protein expression in the two types of chicks fed ad libitum (P < 0.05). These results suggest that insulin resistance exists in the CNS of broiler chicks, possibly due to persistent hyperinsulinemia, which results in a down-regulation of CNS insulin receptor expression compared to that in layer chicks.