Purification of proteins of the Na/Cl cotransporter from membranes of Ehrlich ascites cells using a bumetanide-sepharose affinity column

Summary Bumetanide-binding proteins were isolated from membranes of Ehrlich ascites tumor cells by affinity chromatography. An affinity column was constructed with the active moiety of bumetanide as a ligand using 4-azidobumetanide, a photoactive analogue which inhibits Na/Cl cotransport in Ehrlich cells with high specificity. Covalent binding of the 4-azidobumetanide with Sepharose was promoted by photolysis. Membranes isolated from Ehrlich cells were solubilized withn-octylglucoside. Solubilized proteins retarded by the affinity column were readily eluted by bumetanide. In reducing gels the major proteins eluted by bumetanide were 76 kDa and 38–39 kDa. There were also two proteins of 32 to 35 kDa eluted in lesser amounts. No proteins retarded by the affinity column were eluted with extensive washing without bumetanide. Furthermore, bumetanide eluted no proteins from a control column lacking the specific ligand. Upon rechromatography with bumetanide in solution, bumetanide-eluted proteins were not retarded, but their purity was increased by the retardation of contaminating proteins. Bumetanide-binding protein purified in this manner were characterized further by electrophoresis in nonreducing, nondenaturing gels.     

Gastric lesions produced by conditioned emotional stimuli in the form of affective communication and effects of benzodiazepines

Formation of gastric lesions in response to conditioned emotional stimulus (CES) and effects of benzodiazepines were studied in mice. The CES was introduced in the form of affective communication through a communication box. The "senders" were exposed to electric foot shock and the "responders" were able to receive affective cues such as visual, auditory and olfactory from "senders". The "senders" and "responders" exhibited significantly greater gastric lesions than the controls. Diazepam at doses of 2 X 1 - 2 mg/kg (p.o.) and oxazolam at a dose of 2 X 2 mg/kg (p.o.), reduced the formation of gastric lesions of the "responders" induced non-physically by CES. The present results indicate that "responders" showed bodily changes under CES treatment, particularly in the development of gastric lesions, and that gastric lesions produced by CES were protected by diazepam and oxazolam.     

Glutathione S-transferase in human organs

Glutathione S-transferase (GSH-T) distribution has been investigated in human tissues. The relative contribution of each species to total enzyme activity of the various tissues has been compared. "Cationic" (pI greater than 7.5) "neutral" (pI 6-6.5) and "anionic" (pI less than 5.4) species of GSH-T were separated by isoelectric focusing. "Cationic" GSH-Ts (ligandin) quantitated by radioimmunoassay were present in all tissues studied. Highest concentrations were in liver, kidney, duodenum, testis and adrenal. "Neutral" and "anionic" GSH-Ts were not present in every tissue or in every specimen of some tissues studied. Marked inter-organ and inter-individual variation in the relative concentration of the 3 GSH-T species may explain individual and organ susceptibility to drugs and toxins and underlines the need for future studies to examine individual enzymes rather than total activity.     

Alkaline phosphatase in Amoeba proteus

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.     

Extracellular matrix vesicle distribution in primary mineralization two weeks after injury to rat tibial bone (ultrastructural tissue morphometry)

Primary mineralization on the 14th day of bone healing served as a model to study the distribution of extracellular matrix vesicles by means of transmission electron microscopy combined with computerized morphometry. Vesicles were traced on electron micrographs and classified according to diameter, distance from the calcified front, and type. The different types were determined as follows: electron-lucent vesicles ("empty"), vesicles with amorphous contents ("amorphic"), vesicles containing crystalline depositions ("crystal"), and vesicles with crystals and ruptured membranes ("rupture"). The majority of the vesicles measured between 0.02 and 0.07 micron and were located at a distance of less than 3 micron from the calcified front. They were distributed according to "empty", "amorphic", "crystal" and "rupture" type in concentrations of 10%, 31%, 51% and 8%, respectively. The diameters of the "rupture" vesicles were significantly larger than those of the "empty" and "amorphic" types. The sequence of their location, starting at the calcified front, ran as follows: "rupture", "crystal", "amorphic" and "empty", with the "rupture" type proximate to the front. According to the working hypothesis on calcification via extracellular matrix vesicles, it is thought that the "empty" vesicles are secreted by the cell and that subsequently amorphous Ca and Pi accumulate intravesicularly to form a hydroxyapatite crystal which, in turn, brings about rupture of the vesicle's membrane. The results of the present study support this theory and, additionally, show that the maturation process is accompanied by an increase of the vesicular diameter and by its approximation to the calcifying front.     

cDNA array analysis of alterations in gene expression in the promyelocytic leukemia cell line,HL-60, after apoptosis induction with etoposide

Alterations in gene expression during apoptosis in HL-60 cells were identified by a cDNA based array analysis. Apoptosis was induced in the human promyelocytic leukemia cell line, HL-60, by incubation with 30 M etoposide for 5 hours. Changes in gene expression occurring during apoptosis in these cells were detected using the ATLAS cDNA Expression Array technique. 40 genes were identified as differentially expressed in the apoptotic cells by at least a factor of two. 30 of these genes were down-regulated during apoptosis. Many of the down-regulated genes reflected decreased proliferative activity in the cells as well as decreased activity of survival pathways. Most of the genes, which were up-regulated during apoptosis, were genes involved in pathways leading to cell death and suppression of proliferation. Based on the up-regulations observed at the mRNA level, it is speculated that etoposide-induced apoptosis in the HL-60 cells proceeds via pathways involving factors such as TNF, IGFBP3, SAPK1, AP-1 and GADD153/CHOP10. Four genes, which showed changes at the mRNA level, were also analyzed by Western blotting in order to confirm the observed differences at the protein level.     

Resolution of pea legumin subunits by high-performance liquid chromatography

Pea legumin was dissociated into its component subunits by 6 M urea: these were subsequently fractionated by FPLC using a combination of Mono P, Mono Q, and Mono S columns. The resolution and speed of separation were greatly improved in comparison with previous fractionations. Twelve discrete fractions were obtained and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six "normal" legumin subunits (Mr 60,000) were identified as well as some "large" (Mr 66,000) and "small" (Mr 44,000) subunits. A few polypeptides of unknown origin were also observed. Four subunits were purified to homogeneity as adjudged by electrophoresis and HPLC and in sufficient yields to permit further studies. Anomalous electrophoretic behavior of the legumin subunits was also observed.     

电针对老年性痴呆大鼠外侧隔核AChE阳性神经元表达影响

目的研究电针对老年性痴呆（AD）模型大鼠外侧隔核（LSN）乙酰胆碱酯酶（AChE）阳性神经元表达的影响。方法以D-半乳糖腹腔注射和Aβ1-40海马注射诱导形成老年性痴呆大鼠模型,电针＂大椎＂、＂肾俞＂、＂太溪＂三穴,以免疫组织化学法检测大鼠LSN中AChE阳性神经元的表达。结果与模型组相比,电针组AChE阳性神经元数量明显增多,具有显著统计学意义。结论电针可能通过增加大鼠LSN中AChE阳性神经元的表达,进而发挥抗老年性痴呆的作用。     

Protein kinase C expression and subcellular distribution in chronic myocardial ischemia: Comparison of two different canine models

We studied protein kinase C (PKC) isozyme expression and activity distribution in two models of chronically ischemic canine myocardium: (1) single vessel obstruction (SVO), produced by tight stenosis of LAD followed by preconditioning and acute ischemia (40 min); (2) three vessel obstruction (3VO), produced by LAD-stenosis and gradual occlusion of right coronary artery and left circumflex. In both models after 8 weeks of chronic ischemia the dogs were either sacrificed or had PTCA of the LAD with a follow up of another 4 weeks. Control dogs were sham operated. PKC activity was measured in subcellular fractions of tissue samples from anterior and posterior regions in the presence of histone and -[³²P]-ATP. PKC isozymes were detected by Western blotting. All regions perfused by the obstructed coronaries were dysfunctional at 8 weeks when compared to baseline, with improvement of anterior wall function after PTCA of LAD. PKC activity was elevated in the membrane fraction of SVO, but unchanged in the 3VO model. PKCs , , and prevailed in cytosol fraction of the controls (cytosol/membrane ratios were ± 3.34, 1.38 and 4.56 for , and , respectively), consistent with PKC activity distribution, while was not detected. There was no significant difference between the groups concerning the relative membrane amount of the isozymes. PKCs and were decreased in the cytosol fraction of both models at 8 weeks (for anterior region, by 56 and 57% in SVO and by 49 and 46% in 3VO, respectively) without there being any differences between anterior and posterior regions, and were low also in the PTCA group. PKC distribution however varied between the two models. The amount of PKC isozyme was downregulated by 45% after 8 weeks of chronic ischemia and returned towards the control values after PTCA in the anterior region of SVO, while it did not change in anterior wall after 8 weeks in 3VO but was significantly decreased (by 47%) in posterior region after PTCA. In conclusion, our results suggest modified PKC signalling in chronically ischemic canine myocardium.     

Effects of acute stress on lateralization of lipid peroxidation in the brain depend on the behavioral typology of rats

Using the "emotional resonance test" albino mongrel male rats were separated for their preference to light or dark space during the cry of a "victim" rat receiving footshock. Rats avoiding the cry of a "victim" by moving to the light space were characterized by higher levels of lipid peroxidation (LPO) products. A short footshock resulted in a dramatic LPO decrease in both hemispheres in the brain of all rats under study, but in rats not avoiding the cry of a "victim" (preferring dark space) "left" asymmetry of LPO increased.     

The interrelationship among achievement patterns of activities of daily living for institutionalized dependent elderly

The purpose of the present study was to examine the interrelationship of achievement patterns between 74 activities by selecting nine ADL domains, including 27 items used in existing ADL index for the institutionalized disabled elderly (Disabled-ADL). For these 74 ADL items, 706 institutionalized dependent elderly were assessed using a dichotomous scale of "possible" or "impossible" by staff working at the subjects' institutions. The difficulty of each item was examined by calculating the proportion of "possible" responses. The interrelationships among activities were determined by calculating phi coefficients. Furthermore, the degree of agreement assessed between ADL items was calculated by dividing the total response by the number of "possible-possible" and "impossible-impossible" responses, in order to examine the similarity of the achievement pattern of ADL. The phi coefficient values were high among ADL items with comparable difficulty where the item proportions were within about 10%. Even if belonging to a different ADL domain, the relationship was high among ADL items with similar difficulty and kinds of activity. All of the 27 activities used in Disabled-ADL indicated high agreement of over 80% with one or more activities among the other 47 activities used in this study. Especially, three lower extremity activities of "putting on trousers while standing", "going up and down stairs" and "squatting down from a standing posture" agreed more than 90% with many other lower extremity activities. The possibility is suggested in determining lower extremity functional levels by assessing these three activities. The selection of ADL items considering these influences is necessary to comprehensively assess ADL ability of disabled elderly.     

Influence of thyroid hormone on glycogen metabolism in rat liver.

The livers removed from thyroidectomized and L-T4 supplemented rats were rapidly frozen by Freon-12 chilled with liquid nitrogen, and concentrations of metabolites which affect glycogen synthetase and phosphorylase were determined. Serum and liver glycose levels were not changed in any thyroid functioning. But liver G6P and ATP were increased by thyroidectomy and decreased by L-T4 supplement, while cAMP was increased by the hormone supplement. The "enzyme activity" ratio of glycogen synthetase a to phosphorylase a was increased by thyroidectomy and decreased by L-T4 supplement. The most intimate correlation was observed between the "enzyme activity" ratio and the ratio of the "energy charge" ratio of cAMP among other indices calculated from changes in the metabolite concentrations in the various thyroid functioning. The change in the substrate levels brought about by thyroidectomy and L-T4 supplement appeared to modulate both the enzyme activities which in turn regulate the glycogen metabolism.     

Mechanisms behind active trends in body size evolution of the Canidae (Carnivora: Mammalia)

Clade-level interactions can impart trends on observed characters. In this study, origination and extinction events were compiled for the three subfamilies of the Canidae (dogs): Hesperocyoninae, Borophaginae, and Caninae. These events were binned into 2-million-year time slices, and median body masses were calculated for each time slice. Originations and extinctions were classified as "large" or "small" relative to these median values. Likelihood ratios demonstrate that originations were significantly biased from random in four time slices, while extinctions were never significantly different from random. Two models were chosen by the Akaike Information Criterion to describe the evolutionary dynamics of canid body size. Both proposed a consistent, significant bias toward larger originations, punctuated by episodic "pulses" of increased bias toward larger originations and a Quaternary "pulse" favoring smaller originations, tracking the Quaternary diversification of foxlike canids. These pulses correspond in each subfamily to periods of rapid taxonomic diversification and the appearances of morphologies associated with hypercarnivorous diets. Together, the diversity data, the appearance of hypercarnivory, and the biases in originations document competitive interactions between Hesperocyoninae and Borophaginae and suggest that body size trends were driven, in part, by clade-level dynamics in the Canidae.     

Detergent sensitivity and splitting of isolated liver gap junctions

Summary Isolated rat liver gap junctions were split by two methods. In the first method, isolated gap junctions were stabilized by cross-linking their cytoplasmic surfaces with glutaraldehyde under conditions that prevented the entry of glutaraldehyde into the gap region. The stabilized junctions were then split in the junctional gap with SDS. In the second procedure, unfixed gap junctions were split by incubation in ureacontaining solutions. Junctional splitting was monitored by electron microscopy of thin sectioned and freeze fractured membrane pellets. Sidedness of the split junctional membranes was defined by labeling their cytoplasmic surfaces with glutaraldehyde-activated ferritin before splitting with urea. Gap junctional splitting did not result in any loss of protein components as determined by SDS-gel electrophoresis. The glutaraldehyde cross-linking procedure was also used to determine the effects of various detergents on the protein-protein interactions in the gap region. Of the detergents tested, only SDS caused junctional splitting.     

Characterization of cellulolytic enzyme complexes obtained from mutants of Trichoderma reesei with enhanced cellulase production

Summary The cellulolytic enzyme complexes secreted by the fungus Trichoderma reesei QM 9414 and its mutants M 5, M 6, MHC 15, and MHC 22 were characterized by determining their specific filter-paper (FP)-, carboxymethylcellulase (C_x)-and -glucosidase (G)-activities. They were characterised further by measuring their C_x and G profiles after separation on an isoelectrofocusing column over the pH range 3–10. While the overall FP-activity was roughly equal in all preparations, the specific -glucosidase activity was highest in mutants MHC 15 and MHC 22 which are distingiushed morphologically from the parent strain, QM 9414, by a higher degree of branching of their hyphae. Two peaks of -glucosidase activity were detected by isoelectric focusing in preparations from QM 9414 and M 6, none in the enzyme from the mutant M 5 while 3 and 4 peaks respectively were found in preparations from morphological mutants MHC 15 and MHC 22. The higher -glucosidase activity in these last two preparations was also reflected in the higher glucose to cellobiose ratio in the initial stages of cellulose hydrolysis by the individual enzyme preparations.     

Sodium-23 and potassium-39 nuclear magnetic resonance relaxation in eye lens. Examples of quadrupole ion magnetic relaxation in a crowded protein environment.

Single and multiple quantum nuclear magnetic resonance (NMR) spectroscopic techniques were used to investigate the motional dynamics of sodium and potassium ions in concentrated protein solution, represented in this study by cortical and nuclear bovine lens tissue homogenates. Both ions displayed homogeneous biexponential magnetic relaxation behavior. Furthermore, the NMR relaxation behavior of these ions in lens homogenates was consistent either with a model that assumed the occurrence of two predominant ionic populations, "free" and "bound," in fast exchange with each other or with a model that assumed an asymmetric Gaussian distribution of correlation times. Regardless of the model employed, both ions were found to occur in a predominantly "free" or "unbound" rapidly reorienting state. The fraction of "bound" 23Na+, assuming a discrete two-site model, was approximately 0.006 and 0.017 for cortical and nuclear homogenates, respectively. Corresponding values for 39K+ were 0.003 and 0.007, respectively. Estimated values for the fraction of "bound" 23Na+ or 39K+ obtained from the distribution model (tau C greater than omega L-1) were less than or equal to 0.05 for all cases examined. The correlation times of the "bound" ions, derived using either a two-site or distribution model, yielded values that were at least one order of magnitude smaller than the reorientational motion of the constituent lens proteins. This observation implies that the apparent correlation time for ion binding is dominated by processes other than protein reorientational motion, most likely fast exchange between "free" and "bound" environments. The results of NMR visibility studies were consistent with the above findings, in agreement with other studies performed by non-NMR methods. These studies, in combination with those presented in the literature, suggest that the most likely role for sodium and potassium ions in the lens appears to be the regulation of cell volume by affecting the intralenticular water chemical potential.     

Physiological effects of Shinrin-yoku (taking in the atmosphere of the forest) in an old-growth broadleaf forest in Yamagata Prefecture, Japan

The physiological effects of "Shinrin-yoku" (taking in the atmosphere of the forest) were examined by investigating blood pressure, pulse rate, heart rate variability (HRV), salivary cortisol concentration, and immunoglobulin A concentration in saliva. Subjective feelings of being "comfortable", "calm", and "refreshed" were also assessed by questionnaire. The subjects were 12 male university students aged from 21 to 23 (mean+/-SD: 22.0+/-1.0). The physiological measurements were conducted six times, i.e., in the morning and evening before meals at the place of accommodation, before and after the subjects walked a predetermined course in the forest and city areas for 15 minutes, and before and after they sat still on a chair watching the scenery in the respective areas for 15 minutes. The findings were as follows. In the forest area compared to the city area, 1) blood pressure and pulse rate were significantly lower, and 2) the power of the HF component of the HRV tended to be higher and LF/(LF+HF) tended to be lower. Also, 3) salivary cortisol concentration was significantly lower in the forest area. These physiological responses suggest that sympathetic nervous activity was suppressed and parasympathetic nervous activity was enhanced in the forest area, and that "Shinrin-yoku" reduced stress levels. In the subjective evaluation, 4) "comfortable", "calm", and "refreshed" feelings were significantly higher in the forest area. The present study has, by conducting physiological investigations with subjective evaluations as supporting evidence, demonstrated the relaxing and stress-relieving effects of "Shinrin-yoku".     

Taxonomic Study of Dimocarpus longan by Random Amplified Polymorphic DNA Technique

The classification and relationship among 35 samples of longan (Dimocarpus longan Lour. ) have been studied by RAPD (random amplified polymorphic DNA) and statistical cluster analysis. These results showed that it was unlikely possible that "Lizhiben" and "Lizhi kongyan" were hybrid progeny of longan and litchi ( Litchi chinensis Sonn. ) as what some people believe. The "Lizhiben", and "Nanhu Jiaohe" were highly mutant varieties. There were some variance among the "Jiaohe" wilted-nut longan cultivars. Different cuhivars given the same name, such as "Dongbf', "Honghezi" and some other cultivar, were also possible. It was concluded that longan cultivars which have been tested in this research could be classified into 6 groups. The first group, including 12 cultivars such as "Shuizhang", "Youtanben", "Wulongling" etc., which were the well known best cultivars appropriate for producing dry longan, was named as Shuizhang-Wulongling group. The second group, including 11 cultivars like "Chike", "Jiaoyan", "Fuyan", "Chushuben" etc., was named as Chike-Chushuben group. The Dongbi series cuhivars were named after their cultivars as the Dongbi group. The special cuhivars, "Lidongben", "Nanhu Jiaohe" and "Lizhiben" were named respectively as Lidongben group, Nanhu Jiaohe group, and Lizhiben group, because of their more characteristic DNA polymorphism tested in all respective cuhivars. The results indicate that the application of RAPD technique in the taxonomic study has gained some resolution which could not be obtained by morphological analysis and has provided available data for developing longan production and for breeding new longan cultivars.