Immunohistochemical study of basement membrane reconstruction by an epidermis-dermis recombination experiment using cultured chick embryonic skin: induction of tenascin.

The production of extracellular matrix components such as laminin, Type IV collagen, fibronectin, and tenascin during the formation of basement membrane in cultured epidermis-dermis recombinant skin of 13-day-old chick embryo was analyzed immunohistochemically. The epidermis and dermis were separated from each other by treatment with EDTA and/or dispase. The basal lamina of the basement membrane was thus removed from both epidermis and dermis. The isolated epidermis was overlaid onto the isolated dermis, i.e., recombined, and then cultured for 1-7 days in a chemically defined medium (BGJb) on a Millipore filter. Immunofluorescence labeling was used for light microscopy and HRP or colloidal gold labeling for electron microscopy. In specimens from 2-day cultures, positive sites of anti-laminin and anti-fibronectin reaction were observed light microscopically as patches which, at the electron microscopic level, corresponded to fragments of the basal lamina located immediately beneath and in the vicinity of the attachment plaques of the hemidesmosomes. The staining pattern became continuous 7 days after recombination. Fluorescence labeling of laminin and fibronectin appeared somewhat earlier than that of Type IV collagen and tenascin. All of the four components were found localized primarily in the basal lamina. Furthermore, fibronectin and tenascin were also distributed in the extracellular matrix of the dermis. The expression of tenascin, which does not exist in the basement membrane of 13-day-old intact embryonic skin, was induced in vitro. These results suggest that hemidesmosomes may play an important role in the reconstruction of the basement membrane and that various components of the basement membrane appeared at different times during the reconstruction.     

Inhibition of in vitro cholesterol synthesis by fatty acids.

Inhibitory effect of 44 species of fatty acids on cholesterol synthesis has been examined with a rat liver enzyme system. In the case of saturated fatty acids, the inhibitory activity increased with chain length to a maximum at 11 to 14 carbons, after which activity decreased rapidly. The inhibition increased with the degree of unsaturation of fatty acids. Introduction of a hydroxy group at the alpha-position of fatty acids abolished the inhibition, while the inhibition was enhanced by the presence of a hydroxy group located in an intermediate position of the chain. Branched chain fatty acids having a methyl group at the terminal showed much higher activity than the corresponding saturated straight chain fatty acids with the same number of carbons. With respect to the mechanism for inhibition, tridecanoate was found to inhibit acetoacetyl-CoA thiolase specifically without affecting the other reaction steps in the cholesterol synthetic pathway. The highly unsaturated fatty acids, arachidonate and linoleate, were specific inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA synthase. On the other hand, ricinoleate (hydroxy acid) and phytanate (branched-chain acid) diminished the conversion of mevalonate to sterols by inhibiting a step or steps between squalene and lanosterol.     

Use of ryanodine for functional removal of the calcium store in smooth muscle cells of the guinea-pig

Calcium store of the skinned fibers of the guinea-pig portal vein, pulmonary artery and taenia caeci consisted of two classes: one with both Ca-induced Ca release (CICR) and inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanisms (S alpha) and the other only with IICR mechanisms (S beta). Ryanodine, applied during the CICR was activated, locked the CICR channels open, but the drug had practically no effect on the IICR mechanism. Thus, after the ryanodine treatment the Ca store with the CICR (S alpha) lost its capacity to hold Ca. Changes in the agonist-evoked contraction of intact muscle due to the ryanodine treatment suggest that agonists release Ca from S alpha which produces the initial phase of contractures.     

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol

Mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3α-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD⁺-dependent 3α-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3α-hydroxysteroids and bile acids using NAD⁺ as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3α-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD⁺-dependent type of cytosolic 3α-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3α-HSD resulted in a switch in favor of NADP⁺ specificity, suggesting their key roles in coenzyme specificity.     

Are Additional Trace Elements Necessary in Total Parenteral Nutrition for Patients with Esophageal Cancer Receiving Cisplatin-Based Chemotherapy?

It is known that cisplatin induces the excretion of zinc from the urine and thereby reduces its serum concentration. However, the fluctuation of these trace elements during or after cisplatin-based chemotherapy has not been evaluated. To answer this question, we performed a clinical study in esophageal cancer patients undergoing cisplatin-based chemotherapy. Eighteen patients with esophageal cancer who were not able to swallow food or water orally due to complete stenosis of the esophagus were evaluated. The patients were divided into a control group [total parenteral nutrition (TPN) alone for 28?days, ten cases] and an intervention group (TPN with additional trace elements for 28?days, eight cases). The serum concentrations of zinc, iron, copper, manganese, triiodothyronin (T3), and thyroxin (T4), as alternative indicators of iodine, were measured on days?0, 14, and 28 of treatment, and statistically analyzed on day?28. In the control group, the serum concentration of copper was significantly decreased from 135.4 (day?0) to 122.1???g/ml (day?14), and finally to 110.6???g/ml (day?28, p?=?0.015). The concentration of manganese was also significantly decreased from 1.34 (day?0) to 1.17???g/ml (day?14) and finally to 1.20 (day?28, p?=?0.049). The levels of zinc, iron, T3, and T4 were not significantly changed. In the intervention group, the supplementation with trace elements successfully prevented these decreases in their concentrations. TPN with supplementary trace elements is preferable and recommended for patients who are undergoing chemotherapy in order to maintain the patients?? nutrient homeostasis.     

Gamma-ray exposure from neutron-induced radionuclides in soil in Hiroshima and Nagasaki based on DS02 calculations

As a result of joint efforts by Japanese, US and German scientists, the Dosimetry System 2002 (DS02) was developed as a new dosimetry system, to evaluate individual radiation dose to atomic bomb survivors in Hiroshima and Nagasaki. Although the atomic bomb radiation consisted of initial radiation and residual radiation, only initial radiation was reevaluated in DS02 because, for most survivors in the life span study group, the residual dose was negligible compared to the initial dose. It was reported, however, that there were individuals who entered the city at the early stage after the explosion and experienced hemorrhage, diarrhea, etc., which were symptoms of acute radiation syndrome. In this study, external exposure due to radionuclides induced in soil by atomic bomb neutrons was reevaluated based on DS02 calculations, as a function of both the distance from the hypocenters and the elapsed time after the explosions. As a result, exposure rates of 6 and 4 Gy h(-1) were estimated at the hypocenter at 1 min after the explosion in Hiroshima and Nagasaki, respectively. These exposure rates decreased rapidly by a factor of 1,000 1 day later, and by a factor of 1 million 1 week later. Maximum cumulative exposure from the time of explosion was 1.2 and 0.6 Gy at the hypocenters in Hiroshima and Nagasaki, respectively. Induced radiation decreased also with distance from the hypocenters, by a factor of about 10 at 500 m and a factor of three to four hundreds at 1,000 m. Consequently, a significant exposure due to induced radiation is considered feasible to those who entered the area closer to a distance of 1,000 m from the hypocenters, within one week after the bombing.