Improvement of Transdermal Delivery of Tetragastrin by Lipophilic Modification with Fatty Acids

The in-vitro permeability of chemically modified tetragastrin with fatty acids through the rat skin was studied. The permeability of these compounds through intact skin and stripped skin of rat was determined with a Franz-type diffusion cell. The permeation of tetragastrin across the intact skin was improved by chemical modification with acetic acid and butyric acid. However, tetragastrin and caproyl-tetragastrin did not permeate across the intact skin up to the end of experiment. The permeation of tetragastrin across the stripped skin was improved by chemical modification, the skin flux of these acyl derivatives being in the order: acetyl > butyroyl > caproyl. The stability of tetragastrin in skin homogenate was also significantly improved by chemical modification with fatty acids. These results suggest that chemical modification of tetragastrin with fatty acids increases its lipophilicity, which makes it permeable across the stratum corneum. Moreover, the chemical modification reduced the degradation of tetragastrin in the viable skin, resulting an increase in permeation of tetragastrin across the skin.     

Sequential changes of [3H]cyclic AMP binding in the gerbil brain following transient cerebral ischaemia

Abstract— Sequential alterations in the binding of [³H]cyclic AMP (cAMP) as an indicator of cAMP-dependent protein kinase (cAMP-DPK) binding activity following transient cerebral ischaemia were studied in the gerbil brain using receptor autoradiography. Transient ischaemia was induced for 10 min. [³H]cAMP binding in the stratum oriens and pyramidale of the hippocampal CA1 sector significantly decreased in the early post-ischaemic stage and showed severe reduction 7 days and 1 month after recirculation. By contrast, [³H]cAMP binding showed no significant alterations in the stratum radiatum of the hippocampal CA1 sector and the stratum pyramidale of the hippocampal CA3 sector up to 48 h after ischaemia. However, the binding in these areas significantly decreased 7 days and 1 month after ischaemia. The stratum lacunosum-moleculare of the hippocampal CA1 sector and dentate gyrus showed no significant changes in [³H]cAMP binding throughout the recirculation period. However, in the dorsolateral part of the striatum, where severe neuronal damage was seen morphologically, [³H]cAMP binding was significantly reduced only one month after ischaemia. These results indicate that marked alteration of intracellular signal transduction precedes neuronal damage in the hippocampal CA1 sector, but not in the striatum. Furthermore, our autoradiographic data suggest that post-ischaemic alteration in [³H]cAMP binding between the hippocampal CA1 sector and striatum may be produced by different mechanisms.     

Brain Death in Pediatric Patients in Japan: Diagnosis and Unresolved Issues

Brain death (BD) is a physiological state defined as complete and irreversible loss of brain function. Organ transplantation from a patient with BD is controversial in Japan because there are two classifications of BD: legal BD in which the organs can be donated and general BD in which the organs cannot be donated. The significance of BD in the terminal phase remains in the realm of scientific debate. As indicated by the increasing number of organ transplants from brain-dead donors, certain clinical diagnosis for determining BD in adults is becoming established. However, regardless of whether or not organ transplantation is involved, there are many unresolved issues regarding BD in children. Here, we will discuss the historical background of BD determination in children, pediatric emergencies and BD, and unresolved issues related to pediatric BD.     

Factors Associated with Blunt Cerebrovascular Injury in Patients with Cervical Spine Injury

Blunt cerebrovascular injury (BCVI) is known to be a potentially fatal complication of cervical spine injury (CSI). Methods for screening the appropriate population remain to be elucidated, especially in Japan. This retrospective study was conducted to predict the risk factors relevant to BCVIs. Among 92 patients with CSI transferred to our institution from April 2007 to March 2012, 40 patients (35 men, 5 women) with neurological deficits and/or significant cervical spine fracture including fracture of transversarium, facet, body, lamina, and spinous process, underwent multi-detector computed tomography angiography (MDCTA) and magnetic resonance angiography (MRA), which identified 10 patients with BCVI [2 carotid artery injuries (BCAIs) and 9 vertebral artery injuries (BVAIs); 1 patient suffered both]. Univariate analyses exploring associations between individual risk factors and BCVI and BVAI were performed using Fisher''s exact test and Chi-square test for dichotomous variables and the unpaired t-test for continuous variables. Multiple logistic regression analyses for BCVI and BVAI were carried out using stepwise methods. On univariate and multivariate analysis, hyperextension injury was significantly associated with BVAI (p = 0.01 and p = 0.02), and subluxation (dislocation of vertebral body > 5 mm) was a significant predictor of BCVI (p = 0.04 and p = 0.03) and BVAI (p = 0.01 and p = 0.01). Prompt evaluation for BCVIs is recommended in CSI patients with hyperextension injury and dislocation of the vertebral body.     

Vascular change of hippocampal capillary is associated with vascular change of retinal capillary in aging

Vascular deficiency, such as deleterious change of endothelial cells, becomes the prominent feature of hippocampal microvessels during the processes of aging in rodents and it seems to be associated with deficiency of intellectual behavior in aged subjects. The hippocampal microvessels and hippocampal pyramidal neurons form and accumulate intermediates of advanced Maillard reaction (glycation) end products, specifically N()-carboxymethyl lysine (CML) and CML in rodents during the processes of aging. CML facilitates proliferation of endothelial cells in culture. However, further conjugation of CML with the substance(s) seems to occur in the microvessels and pyramidal neurons of hippocampus and it brings about deleterious change of endothelial cells and pyramidal neuron death. This would cause deficiency of recognition and reference memory in rodents during the processes of aging. In man in Alzheimer's disease (AD), one might speculate that formation and accumulation of CML in the hippocampal microvessels initiate the accumulation of amyloid to produce cerebral amyloid angiopathy and it brings about hypoglycemia and hypoxia in the hippocampal pyramidal neurons. Furthermore, formation and accumulation of CML in the hippocampal pyramidal neurons initiate the deposition of neurofibrillary tangles and senile plaques which cause neuronal death. In this way, vascular deficiency of hippocampal microvessels seems to be associated with the demented disease, the atrophic process of the brain and accumulation of amyloid in the brain in man. In terms of vascular deficiency concerns, the vascular change of the retinal capillaries becomes also a prominent feature during the processes of aging and it has a positive correlation with the vascular change of hippocampal capillary. In man during senescence, one might also speculate that vascular change of eye capillaries would become the early market for diagnosis of dementia in AD.     

Overproduction of N(epsilon)-(carboxymethyl)lysine-induced neovascularization in cultured choroidal explant of streptozotocin-diabetic rat

Action of N(epsilon)-(carboxymethyl)lysine (CML) adduct, an advanced glycation end product, was investigated on neovascularization of cultured choroidal explants in streptozotocin (STZ)-diabetic rat. The choroidal explants of early (4 weeks after an injection of 60 mg/kg STZ) and advanced (8 months after the STZ injection) diabetic rats, and age-matched normal rats were cultured in fibrin gel with Dulbecco's modified Eagle medium containing fetal bovine serum. The number of budded microvessel-like structures was counted and used as an index of in vitro neovascularization. Choroidal explants in the early diabetic stage released vascular endothelial growth factor (VEGF) and tended to increase tumor necrosis factor (TNF) alpha and platelet-derived growth factor (PDGF)-B, and concomitantly facilitated growth of sprout and buds, compared to the normal control. When choroidal explants were stimulated with CML-human serum albumin (HSA), its releasing effect was in the order VEGF>TNFalpha>PDGF-B. CML-HSA and CML-bovine serum albumin augmented the neovascularization in the cultured diabetic explant and their actions did not virtually differ. A monoclonal anti-CML antibody (6D12) inhibited the neovascularization in the advanced diabetes greater than that in the early diabetes. Inhibitory actions of anti-VEGF and anti-TNFalpha antibodies on the neovascularization were similar to that of the anti-CML antibody in the diabetes. In conclusion, CML adducts were accumulated and over-produced the actions of VEGF, TNFalpha and PDGF-B in the choroidal explant during diabetes in an age-dependent manner. TNFalpha and VEGF are likely to play a predominant role for the CML-induced choroidal neovascularization.     

Effect of manoalide on apoptosis induced by deprivation of growth factors in vascular endothelial cells

研究ｍａｎｏａｌｉｄｅ对除去生长因子（ａＦＧＦ和血清）而诱导的血管内皮细胞凋亡的影响．方法：通过细胞形态观察,ＤＮＡ凝胶电泳及荧光显微术等方法确定ｍａｎｏａｌｉｄｅ对细胞凋亡的抑制或促进作用．结果：向去除ａＦＧＦ和血清的培养液中加低浓度的ｍａｎｏａｌｉｄｅ（１－４μｍｏｌ·Ｌ－１）,培养细胞４８ｈ,细胞的脱壁和ＤＮＡ片断化受到抑制;ｍａｎｏａｌｉｄｅ浓度为７μｍｏｌ·Ｌ－１时,促进细胞脱壁和ＤＮＡ片断化．结论：低浓度的ｍａｎｏａｌｉｄｅ（２μｍｏｌ·Ｌ－１）抑制血管内皮细胞凋亡,而较高浓度的ｍａｎｏａｌｉｄｅ（７μｍｏｌ·Ｌ－１）促进该细胞凋亡．     

Combined immunohistochemical study of tissue polypeptide antigen and cancer antigen 125 in human ovarian tumours

An indirect immunoperoxidase method was used to study the expression of tissue polypeptide antigen (TPA) and cancer antigen 125 (CA 125) in 47 benign and malignant ovarian tumours. Tissue polypeptide antigen and CA 125 antigen were expressed respectively in 22 (73%) and 16 (53%) of the 30 adenocarcinomas and in five (29%) and four (23%) of the 17 benign tumours. Co-expression of TPA and CA 125 antigen occurred in 12 (40%) malignant and four (23%) benign tumours. Ultrastructurally, TPA and CA 125 antigens were located at the cell surface and microvillous surfaces. Evaluation of combined TPA and CA 125 antigen results revealed a remarkable improvement in the positivity rate and a significant decrease (P less than 0.05) in the negativity rate of ovarian carcinomas as compared with the result of each one separately. These findings provide complementary evidence for the previous results on the plasma levels of TPA and CA 125 antigen and suggest that specific combinations of tumour markers may be more effective for the diagnosis and monitoring of ovarian carcinomas, than the use of any single marker.     

Effects of volatile anesthetics on the calcium ionophore A23187-mediated alterations in hepatic flow and metabolism in the perfused liver in fasted rats

Alterations in intracellular calcium homeostasis have been implicated in hepatic injury. Volatile anesthetics modulate the homeostasis of intracellular calcium.
The effects of volatile anesthetics on the hemodynamic and metabolic alterations induced by the calcium ionophore A23187 were studied using isolated liver perfusion in fasted rats. The liver was isolated from 24 hr-fasted male Sprague-Dawley rats, and perfused through the portal vein at a constant pressure of 1.2 kPa in a recirculating perfusion-aeration system. Halothane, isoflurane and sevoflurane were administered at 2%, 3% and 4.4%, respectively.
All volatile anesthetics maintained basal hepatic flow, reduced oxygen consumption, and transiently enhanced net lactate production. A23187 at initial concentrations of 0.8 to 3.2 μM decreased hepatic flow and oxygen consumption in a dose-de pendent manner, and enhanced lactate production. All anesthetics significantly attenuated the decreases in hepatic flow and oxygen consumption after administration of A23187 at 1.6 μM. None of the anesthetics significantly influenced the A23187-induced enhancement of net lactate production.
Volatile anesthetics may attenuate the hepatic vasoconstriction and oxygen debt induced by intracellular calcium overload.     

Glyoxal inactivates glutamate transporter‐1 in cultured rat astrocytes

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor paralysis and selective motor neuron death. There is increasing evidence that motor neuron death in ALS is mediated by glutamate toxicity resulting from reduced activity of astrocytic glutamate transporter‐1 (GLT‐1). Recent morphological studies have shown that N^?‐(carboxymethyl)lysine (CML) accumulates in reactive astrocytes of ALS spinal cords. CML is a product of post‐translational protein modification by glyoxal, a reactive aldehydic intermediate. In considering these documents, it is important to determine whether GLT‐1 protein modification by glyoxal might cause reduced GLT‐1 activity. To address this issue, we investigated the effects of glyoxal on GLT‐1 properties in cultured rat astrocytes. High performance liquid chromatography showed reduced glutamate uptake activity in the glyoxal‐exposed cells. Immunocytochemical analysis displayed CML accumulation in the cytoplasm of astrocytes by glyoxal exposure. Immunoblots of immunoprecipitated GLT‐1 disclosed GLT‐1 CML adduct formation in the glyoxal‐exposed cells. Our results indicate that glyoxal modifies GLT‐1 to form CML and simultaneously deprives its glutamate uptake activity. Thus, these toxic effects of glyoxal on astrocytes might be implicated in motor neuron death in ALS.